Constructs for expressing lysomal polypeptides

ABSTRACT

Provided are isolated nucleic acids for expressing lysosomal polypeptides such as lysosomal acid α-glucosidase (GAA) and vectors comprising the same. In one embodiment, the invention provides an isolated nucleic acid encoding a chimeric polypeptide comprising a secretory signal sequence operably linked to a lysosomal polypeptide. In another representative embodiment, an isolated nucleic acid is provided comprising a coding region encoding a GAA and a GAA 3′ untranslated region (UTR), wherein the GAA 3′ UTR comprises a deletion therein. In another representative embodiment, the invention provides an isolated nucleic acid comprising a coding region encoding a GAA and a 3′ UTR, wherein the 3′ UTR is less than about 200 nucleotides in length and comprises a segment that is heterologous to the GAA coding region. Also provided are methods of making and using delivery vectors encoding lysosomal polypeptides, for example, to produce the lysosomal polypeptide or to treat subjects afflicted with a deficiency in the lysosomal polypeptide.

RELATED APPLICATION INFORMATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/441,789, filed Jan. 22, 2003, which is incorporatedby reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to novel nucleic acid constructsencoding lysosomal polypeptides, in particular lysosomal acida-glucosidase, as well as methods of using the same to producerecombinant lysosomal polypeptides and to treat lysosomal polypeptidedeficiencies.

BACKGROUND OF THE INVENTION

[0003] Glycogen storage disease type II (GSD II) is a classicallysosomal storage disorder, characterized by lysosomal accumulation ofglycogen and tissue damage, primarily in muscle and heart (Hirschhorn,R. and Reuser, A. J. (2001) In The Metabolic and Molecular Basis forInherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle,D. Eds.), pp. 3389-3419. McGraw-Hill, New York). The underlying enzymedeficiency is acid a-glucosidase (GAA). In severe, infantile GSD IIprogressive cardiomyopathy and myopathy lead to cardiorespiratoryfailure and death by 1 year of age. In milder, juvenile and adult-onsetGSD II, progressive weakness and respiratory failure are disabling anddeath from respiratory failure occurs.

[0004] Animal models for human lysosomal acid α-glucosidase (hGAA)deficiency accurately mimic GSD II, and the efficacy of approaches togene therapy for GSD II can be evaluated in these systems. The GAAknockout (GAA-KO) mouse model accumulated glycogen in skeletal andcardiac muscle, and developed weakness and reduced mobility (Raben, N.,et al. (1998) J. Biol. Chem. 273:19086-19092, Bijvoet, A. G., et al.(1998) Hum. Mol. Genet. 7:53-62). The administration of recombinant GAAto a GM-KO mouse demonstrated uptake of GAA by skeletal muscle,presumably through receptor-mediated uptake and delivery of GAA to thelysosomes (Bijvoet, A. G. et al. (1998) Hum. Mol. Genet. 7:1815-1824).The Japanese quail model is similar to juvenile-onset GSD II, and hasbeen treated successfully with recombinant enzyme replacement (Kikuchi,T., et al. (1998) J. Clin. Invest. 101:827-833). Enzyme therapy hasdemonstrated efficacy for severe, infantile GSD II; however the benefitof enyzme therapy is limited by the need for frequent infusions and thedevelopment of inhibitor antibodies against recombinant hGAA(Amalfitano, A., et al. (2001) Genet. In Med. 3:132-138). As analternative or adjunct to enzyme therapy, the feasibility of genetherapy approaches to treat GSD-II have been investigated (Amalfitano,A., et al. (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866, Ding, E., etal. (2002) Mol. Ther. 5:436-446, Fraites, T. J., et al. (2002) Mol.Ther. 5:571-578, Tsujino, S., et al. (1998) Hum. Gene Ther.9:1609-1616).

[0005] Administration of an adenovirus (Ad) vector encoding hGAA thatwas targeted to mouse liver in the GAA-KO mouse model reversed theglycogen accumulation in skeletal and cardiac muscle within 12 daysthrough secretion of hGAA from the liver and uptake in other tissues(Amalfitano, A., et al. (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866).Antibodies against hGAA abbreviated the duration of hGAA secretion withan Ad vector in liver, although vector DNA and hGAA persisted in tissuesat reduced levels for many weeks (Ding, E., et al. (2002) Mol. Ther.5:436446). Introduction of adeno-associate virus 2 (AAV2) vectorsencoding GAA normalized the GM activity in the injected skeletal muscleand the injected cardiac muscle, and glycogen content was normalized inmuscle when an AAV1-pseudotyped vector was administered with improvedmuscle transduction (Fraites, T. J., et al. (2002) Mol. Ther.5:571-578). Muscle-targeted Ad vector gene therapy was attempted in theJapanese quail model, although only localized reversal of glycogenaccumulation at the site of vector injection was achieved (Tsujino, S.,et al. (1998) Hum. Gene Ther. 9:1609-1616).

[0006] Neonatal gene therapy may have greater efficacy thanadministration later in life, as evidenced by experiments in severalrodent disease models. An AAV vector administered intravenously on thesecond day of life in β-glucoronidase deficient (Sly disease) miceproduced therapeutically relevant levels of P-glucoronidase andcorrected lysosomal storage in multiple tissues, including liver andkidney (Daly, T. M., et al. (1999) Proc. Natl. Acad. Sci. USA96:2296-2300). Similarly, intramuscular injection of the AAV vectorproduced sustained, therapeutic levels of expression of β-glucoronidaseand eliminated lysosomal storage in muscle and liver (Daly, T. M., etal. (1999) Hum. Gene Ther. 10:85-94). AAV vector DNA persisted in muscleand in transduced areas of the liver following neonatal intramuscularinjection in the Sly disease mouse (Daly, T. M., et al. (1999) Hum. GeneTher. 10:85-94). The number of AAV vector particles administered toneonatal Sly mice was approximately 100-fold less than was needed toproduce therapeutically relevant levels of proteins in adult mice(Kessler, P. D., et al. (1996) Proc. Natl. Acad. Sci. USA93:14082-14087, Herzog, R. W., et al. (1997) Proc. Nat. Acad. Sci. USA94:5804-5809, Nakai, H., et al. (1998) Blood 91:4600-4607, Snyder, R.O., et al. (1997) Nat. Genet. 16:270-276, Koeberl, D. D., et al. (1999)Hum. Gene Ther. 10:2133-2140, Snyder, R. O., et al. (1999) Nat. Med.5:64-70, Herzog, R. W. et al. (1999) Nat. Med. 5:56-63).

[0007] There is a need in the art for improved methods of producinglysosomal polypeptides such as GAA in vitro and in vivo, for example, totreat lysosomal polypeptide deficiencies. Further, there is a need formethods that result in systemic delivery of GAA and other lysosomalpolypeptides to affected tissues and organs.

SUMMARY OF THE INVENTION

[0008] The present invention is based, in part, on the discovery ofimproved nucleic acid constructs for expressing lysosomal polypeptidessuch as GAA. One aspect of the invention provides an isolated nucleicacid encoding a chimeric polypeptide comprising a lysosomal polypeptideoperably linked to a secretory signal sequence such that targeting ofthe lysosomal polypeptide to the secretory pathway (i.e., instead of tothe lysosome) is enhanced. As another aspect, the invention encompassesisolated nucleic acids comprising a coding sequence for GAA and an“abbreviated” 3′ untranslated region (3′ UTR). The isolated nucleicacids of the invention can be used to produce recombinant lysosomalpolypeptides in vitro (e.g., in cultured cells) or in vivo (e.g., in ananimal or plant based protein production system or in methods oftherapeutic treatment). In addition, improved (i.e., higher) titers ofviral vectors encoding lysosomal polypeptides can be produced with theisolated nucleic acids of the invention.

[0009] Accordingly, as one aspect, the present invention provides anisolated nucleic acid encoding a chimeric polypeptide comprising asecretory signal sequence operably linked to a lysosomal polypeptide(e.g., GAA). Also provided is the chimeric polypeptide comprising thesecretory signal sequence operably linked to the lysosomal polypeptide.

[0010] As another aspect, the present invention provides isolatednucleic acids comprising a GAA coding sequence and an “abbreviated” 3′UTR. In one representative embodiment, the invention provides anisolated nucleic acid encoding a GAA, the isolated nucleic acidcomprising: (a) a coding region encoding a GAA, and (b) an abbreviated3′ UTR, wherein the 3′ untranslated region is a GAA 3′ UTR comprising adeletion therein (e.g., a deletion of at least 25 consecutivenucleotides), so that upon introduction into a cell, GAA polypeptide isproduced at a higher level from the isolated nucleic acid as comparedwith GAA polypeptide production from an isolated nucleic acid comprisinga full-length GAA 3′ UTR. In an exemplary embodiment, the 3′ UTRcomprises a deletion in the region shown as nucleotides 3301 through3846 of SEQ ID NO:1 (FIG. 8).

[0011] In another representative embodiment, the invention provides anisolated nucleic acid encoding a GAA, the isolated nucleic acidcomprising: (a) a coding region encoding a GAA, and (b) an abbreviated3′ UTR, wherein the 3′ UTR is less than about 200 nucleotides in lengthand comprises a segment that is heterologous to the GAA coding region,so that upon introduction into a cell, GAA polypeptide is produced at ahigher level from the isolated nucleic acid as compared with GAApolypeptide production from an isolated nucleic acid comprising afull-length GAA 3′ UTR.

[0012] In other particular embodiments, the lysosomal polypeptide is ahuman polypeptide and/or the isolated nucleic acid is operativelyassociated with a transcriptional control element that is operable inliver cells (optionally, with a liver-specific transcriptional controlelement).

[0013] As additional aspects, the present invention further providesvectors (including nonviral and viral vectors, the latter includingadenovirus, AAV and hybrid adenovirus-AAV vectors), cells andpharmaceutical formulations comprising the isolated nucleic acids ofthis invention.

[0014] As still further aspects, the present invention provides methodsof making delivery vectors (e.g., viral vectors) comprising the isolatednucleic acids of this invention.

[0015] Also provided are methods of using the isolated nucleic acids,vectors, cells, and pharmaceutical formulations of the invention totreat deficiencies of lysosomal polypeptides (e.g., GAA) and to producerecombinant lysosomal polypeptides (e.g., in vitro in cultured cells orin vivo in an animal or plant-based recombinant protein expressionsystem or for therapeutic purposes).

[0016] In illustrative embodiments, the present invention is practicedto administer an isolated nucleic acid encoding a lysosomal polypeptidesuch as GAA to a subject (for example, a subject diagnosed with orsuspected of having a deficiency of the lysosomal polypeptide). Inparticular representative embodiments, an isolated nucleic acid of theinvention encoding a lysosomal polypeptide can be administered to onedepot tissue or organ (e.g., liver, skeletal muscle, lung and the like)and the polypeptide expressed therein at levels sufficient to result insecretion into the systemic circulation, from which the secretedpolypeptide is taken up by distal target tissues (e.g., skeletal,cardiac and/or diaphragm muscle). Similarly, the isolated nucleic acidcan be delivered to brain cells (e.g., to treat MPS disorders such asSly disease), where the lysosomal polypeptide can be expressed, secretedand taken up by non-transformed or non-transduced brain cells (e.g.,neurons and glial cells).

[0017] In another particular embodiment, a recombinant adeno-associatedvirus (AAV) vector expressing an isolated nucleic acid encoding GAA ofthe invention is administered to the liver of a subject having GAAdeficiency, which results in GAA polypeptide production at sufficientlyhigh levels such that GAA polypeptide is secreted from the liver andtaken up by skeletal muscle and/or other tissues, which advantageouslyleads to a reduction in glycogen content and/or an improvement in otherclinical indicia of GAA deficiency in affected tissues.

[0018] As yet a further aspect, the present invention provides the useof the isolated nucleic acids, vectors, cells and pharmaceuticalformulations of the invention in the manufacture of medicaments for thetreatment of lysosomal polypeptide deficiencies (e.g., GAA deficiency).

[0019] These and other aspects of the invention are described in moredetail in the description of the invention set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1. Detection of hGAA following neonatal intramuscularadministration of an Ad-AAV vector in GAA-KO mice. (Panel A) Westernblot analysis of muscle after gastrocnemius of GAA-KO mice was injectedwith the Ad-AAV vector (4×10¹⁰ DRP) encoding hGAA at 3 days of age. Allsamples were obtained 24 weeks following vector administration.Recombinant human GAA (rhGAA) was the standard. Each lane for theindicated muscle groups represents one GAA-KO mouse. The ˜67 kD, ˜76 kD,and ˜110 kD hGAA species were detected in transduced muscle as expected(Amalfitano, A., et al. (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866,Ding, E., et al. (2002) Mol. Ther. 5:436-446). (Panel B) Western blotanalysis of heart, liver and diaphragm following neonatal administrationof the Ad-AAV vector. Each lane represents one GAA-KO mouse analyzed atthe indicated time following vector administration. The samples forindividual mice were loaded in the same order for the Western blot ofeach tissue.

[0021]FIG. 2. GAA activity and glycogen content for skeletal muscle andother tissues in GAA-KO mice following neonatal Ad-AAV vectoradministration. (Panel A) The hGAA activity for gastrocnemius andquadriceps at 6, 12, and 24 weeks following vector administration, andfor hamstrings at 24 weeks, compared to the hGAA activity ingastrocnemius, quadriceps, and hamstrings for untreated, GAA-KO mice.The average and standard deviation are shown. The p value is indicatedas follows: *<0.05, **<0.01, and ***<0.001. The number of mice (n) isshown for each time point. (Panel B) The hGAA activity for heart, liver,and diaphragm in mice following Ad-AAV vector administration, and forcontrols. Controls were untreated, GAA-KO mice: n=4 for heart and liver,n=5 for diaphragm. The average and standard deviation are shown forcontrols.

[0022]FIG. 3. Antibodies in GAA-KO mouse plasma following, neonatal,Ad-AAV vector administration. (Panel A) The absorbance for anti-hGAAantibodies in an ELISA of GAA-KO mouse plasma at 6 weeks followingneonatal Ad-AAV vector administration (Neonatal Intramuscular Ad-AAV),and of GAA-KO mouse plasma at 6 weeks following intravenous Ad-AAVvector administration (4×10¹⁰ particles) in adult mice (Ad-AAV).Controls consisted of untreated, GAA-KO mice (Control). Each columnrepresents the mean and standard deviation for an individual mouse. Eachdilution of plasma (1:100, 1:200, and 1:400) was analyzed in triplicate.(Panel B) The titer for anti-hGAA antibodies by ELISA, representing thesame mice in the same order as in FIG. 1 Panel B. Each sample wasanalyzed in duplicate at each dilution. (Panel C) The absorbance foranti-Ad antibodies in an ELISA of GAA-KO mouse plasma for the samesamples as described for the ELISA for anti-hGAA antibodies above,except that only 2 untreated controls were analyzed. Each sample wasanalyzed in duplicate at each dilution. The order of loading was thesame as in FIG. 1 Panel B.

[0023]FIG. 4. The glycogen content for skeletal muscle and heart. (PanelA) Gastrocnemius and quadriceps at 6, 12, and 24 weeks following vectoradministration, and for hamstrings at 24 weeks, compared to the hGAAactivity in gastrocnemius, quadriceps, and hamstrings for untreated,GAA-KO mice. The average and standard deviation are shown. The p valueis indicated as follows: *<0.05 **<0.01 , and *<0.001. The number ofmice (n) is shown for each time point. (Panel B) The glycogen contentfor heart and diaphragm, and for controls. Controls were untreated,GAA-KO mice (n=4). The average and standard deviation are shown forcontrols.

[0024]FIG. 5. Glycogen staining of skeletal muscle and heart. PASstaining showed glycogen accumulation in lysosomes, and pooling ofglycogen outside lysosomes, that was corrected following Ad-AAVadministration at the times indicated.

[0025]FIG. 6. hGAA synthesis (top panel) and glycogen content (lowerpanel) in muscle of GAA-KO/SCID mice administered an AAV2/6 (AAV6)vector expressing GAA intramuscularly.

[0026]FIG. 7. Secretion of hGAA from liver into plasma in GA-KO/SCIDmice administered an AAV2/2 (AAV2) or AAV2/6 (AAV6) vector. Western blotanalysis of plasma from GAA-KO/SCID mice following AAV vectoradministration, and from untreated GAA-KO/SCID mice (controls).

[0027]FIG. 8. A full-length hGAA cDNA sequence; Genebank Accession No.NM_(—)000152 (SEQ ID NO:1). The encoded protein is shown in SEQ ID NO:2.The ORF is nt 442 . . . 3300. The GAA 3′ UTR sequence is from nt 3301 to3846, total 546 bp.

[0028]FIG. 9. A hGAA sequence with a deleted 3′ UTR (SEQ ID NO:3). The411 bp from nt 3397 through to nt 3807 in the 3′ UTR of the sequenceshown in FIG. 8 (SEQ ID NO:1) were deleted (bold, italic). Note that thepolyA signal (bold, 3825 . . . 3830) and polyA site (3846) are notdeleted. The 5′ UTR sequence of nt 1 through nt 409 was also deleted.

[0029]FIG. 10. GAA activity in liver and other tissues following portalvein injection of an AAV2/2 vector. GAA-KO/SCID mice received the vectorpackaged as AAV2 (AAV2/2) at 3 months of age (n=3), and were analyzed 12weeks after injection. Controls were 3 month-old, untreated GAA-KO/SCIDmice (n=3).

[0030]FIG. 11. Human GAA in liver and other tissues following portalvein injection of AAV vectors in GAA-KO/SCID mice. Western blot analysisof plasma from GAA-KO/SCID mice at the indicated times following AAVvector administration, and from untreated GAA-KO/SCID mice (controls).Recombinant hGAA (rhGAA) is shown as a standard.

[0031]FIG. 12. GAA activity in gastrocnemius muscles (injected anduninjected) and other tissues following intramuscular injection of anAAV2/6 vector. GAA-KO/SCID mice received the vector packaged as AAV 6(AAV2/6) at 6 weeks of age (n=8), and were analyzed at 6,12 and 24 weeksafter injection. Controls were 3 month-old, untreated GAA-KO/SCID mice(n=3).

[0032]FIG. 13. Glycogen staining of skeletal muscle in GAA-KO/SCID mice.PAS staining showed glycogen accumulation in lysosomes, and pooling ofglycogen outside lysosomes that was corrected following AAV2/6administration.

[0033]FIG. 14. Localization (cellular vs. secreted) of GAA with variousleader sequences expressed in transfected 293 cells.

[0034]FIG. 15. Western blot analysis of GAA with various leadersequences. GAA expressed in transfected 293 cells with the constructscontaining hGAA linked to the indicated signal peptides. For transfected293 cells, in lanes 3-14, the first (odd-numbered) of 2 lanes for eachconstruct represents the cell lysate and the second lane (even-numbered)is the medium. The control represents untransfected 293 cells, showingendogenous, processed hGAA. Recombinant human GAA (rhGAA) is shown as astandard.

[0035]FIG. 16. Western blot analysis of plasma from GAA-KO/SCID mice at2 weeks following vector administration, and from untreated GAA-KO/SCIDmice (controls). Three-month-old GAA-KO/SCID mice received an AAV vectorencoding the chimeric α-1-antitrypsin signal peptide linked to the hGAAcDNA (Alpha-1-antitrypsin, lanes 2-7), or an AAV vector encoding hGAAwith its endogenous signal peptide (hGAA, lanes 8-13). Each lanerepresents an individual mouse. Lanes 5-7 and 11-13 were female mice.Recombinant human GAA (rhGAA) is shown as a standard.

[0036]FIG. 17. GAA activity in liver and other tissues at 2 weeksfollowing intravenous injection of an AAV2/8 vector encoding hGAA linkedto the alpha-1-antitrypsin leader sequence. Male 3 month-old GAA-KO/SCIDmice (n=3) received an AAV vector encoding the chimericalpha-1-antitrypsin signal peptide linked to the hGAA cDNA (minus the 27amino acid GAA signal peptide). Controls were untreated GAA-KO/SCID mice(n=3).

[0037]FIG. 18. Human GAA in liver and other tissues followingintravenous injection of an AAV vector containing a liver-specificpromoter to drive GAA expression in immunocompetent GAA-KO mice. Westernblot analysis of plasma from GAA-KO mice at 3 weeks following AAV vectoradministration, and from untreated GAA-KO mice (controls). Recombinanthuman rhGAA (rhGAA) is shown as a standard.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The present invention is based, in part, on the discovery ofimproved nucleic acid constructs for expressing lysosomal polypeptidessuch as GAA. One aspect of the invention provides an isolated nucleicacid encoding a chimeric polypeptide comprising a lysosomal polypeptideoperably linked to a secretory signal sequence such that targeting ofthe lysosomal polypeptide to the secretory pathway (i.e., instead of tothe lysosome) is enhanced. As another aspect, the invention encompassesisolated nucleic acids comprising a coding sequence for GAA and an“abbreviated” 3′ UTR.

[0039] The isolated nucleic acids of the invention are advantageous fordelivery of lysosomal polypeptides (e.g., GAA) to target cells or forrecombinant protein production in cultured cells or tissues or wholeanimal systems (e.g., for enzyme replacement therapy). In particularembodiments, the present invention can be practiced to deliver a codingsequence for a lysosomal polypeptide to a “depot” tissue or organ (e.g.,liver, skeletal muscle, lung), where the polypeptide is expressed in thedepot tissue or organ, secreted into the systemic circulation, and takenup by target tissues (e.g., skeletal muscle, cardiac muscle and/ordiaphragm muscle in GAA deficient individuals). In representativeembodiments, uptake of GAA polypeptide secreted from the liver byskeletal muscle and/or other tissues affected by GAA deficiency resultsin a reduction in glycogen stores or improvement in other clinicalindicia of GAA deficiency. In other embodiments, the isolated nucleicacid is delivered to cells (e.g., neurons and/or glial cells) in thebrain, the lysosomal polypeptide is produced and secreted by thetransformed or transduced cells and taken up by other brain cells.

[0040] The present invention will now be described with reference to theaccompanying drawings, in which preferred embodiments of the inventionare shown. The terminology used in the description of the inventionherein is for the purpose of describing particular embodiments only andis not intended to be limiting of the invention. This invention may beembodied in different forms and should not be construed as limited tothe embodiments set forth herein. Rather, these embodiments are providedso that this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Forexample, features illustrated with respect to one embodiment may beincorporated into other embodiments, and features illustrated withrespect to a particular embodiment may be deleted from that embodiment.In addition, numerous variations and additions to the embodimentssuggested herein will be apparent to those skilled in the art in lightof the instant disclosure, which do not depart from the instantinvention.

[0041] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs.

[0042] All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

[0043] Except as otherwise indicated, standard methods may be used forthe production of viral and non-viral vectors, manipulation of nucleicacid sequences, production of transformed cells, recombinant proteinproduction, and the like according to the present invention. Suchtechniques are known to those skilled in the art. See, e.g., SAMBROOK etal., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor,N.Y., 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NewYork).

[0044] Definitions.

[0045] Unless indicated otherwise, explicitly or by context, thefollowing terms are used herein as set forth below:

[0046] As used in the description of the invention and the appendedclaims, the singular forms “a,” “an” and “the” are intended to includethe plural forms as well, unless the context clearly indicatesotherwise.

[0047] As used herein, a “vector” or “delivery vector” may be a viral ornon-viral vector that is used to deliver a nucleic acid to a cell,tissue or subject.

[0048] A “recombinant” vector or delivery vector refers to a viral ornon-viral vector that comprises one or more heterologous nucleotidesequences (i.e., transgenes), e.g., two, three, four, five or moreheterologous nucleotide sequences. The recombinant vectors of theinvention comprise nucleotide sequences that encode GAA, but may alsocomprise one or more additional heterologous sequences.

[0049] As used herein, the term “viral vector” or “viral deliveryvector” may refer to a virus particle that functions as a nucleic aciddelivery vehicle, and which comprises the recombinant vector genomepackaged within a virion. Alternatively, these terms may be used torefer to the vector genome when used as a nucleic acid delivery vehiclein the absence of the virion.

[0050] A viral “vector genome” refers to the viral genomic DNA or RNA,in either its naturally occurring or modified form. A “recombinantvector genome” is a viral genome (e.g., vDNA) that comprises one or moreheterologous nucleotide sequence(s).

[0051] A “heterologous nucleotide sequence” will typically be a sequencethat is not naturally-occurring in the vector. Alternatively, aheterologous nucleotide sequence may refer to a sequence that is placedinto a non-naturally occurring environment (e.g., by association with apromoter with which it is not naturally associated).

[0052] By “infectious,” as used herein, it is meant that a virus canenter a cell by natural transduction mechanisms and express viral genes(including heterologous nucleotide sequence(s)). Alternatively, an“infectious” virus is one that can enter the cell by other mechanismsand express the genes encoded by the viral genome. As one illustrativeexample, the vector can enter a target cell by expressing a ligand orbinding protein for a cell-surface receptor in the virion or by using anantibody(ies) directed against molecules on the cell-surface followed byinternalization of the complex.

[0053] As used herein, “transduction” of a cell by AAV means that theAAV enters the cell to establish a latent infection. See, e.g., BERNARDN. FIELDS et al., VIROLOGY, volume 2, chapter 69.(3d ed.,Lippincott-Raven Publishers).

[0054] The term “replication” as used herein in reference to viralvectors refers specifically to replication (i.e., making new copies of)of the vector genome (i.e., virion DNA or RNA).

[0055] The term “propagation” as used herein in reference to viralvectors refers to a productive viral infection wherein the viral genomeis replicated and packaged to produce new virions, which typically can“spread” by infection of cells beyond the initially infected cell. A“propagation-defective” virus is impaired in its ability to produce aproductive viral infection and spread beyond the initially infectedcell.

[0056] As used herein, the term “polypeptide” encompasses both peptidesand proteins, unless indicated otherwise.

[0057] A “chimeric polypeptide” is a polypeptide produced when twoheterologous genes or fragments thereof coding for two (or more)different polypeptides or fragments thereof not found fused together innature are fused together in the correct translational reading frame.Illustrative chimeric polypeptides include fusions of GAA or otherlysosomal polypeptides to all or a portion of glutathione-S-transferase,maltose-binding protein, or a reporter protein (e.g., Green FluorescentProtein, β-glucuronidase, and β-galactosidase, luciferase). Inparticular embodiments of the invention, the chimeric polypeptidecomprises a secretory signal sequence operably linked to a lysosomalpolypeptide (e.g., GAA).

[0058] As used herein, a “functional” polypeptide is-one that retains atleast one biological activity normally associated with that polypeptide.Preferably, a “functional” polypeptide retains all of the activitiespossessed by the unmodified polypeptide. By “retains” biologicalactivity, it is meant that the polypeptide retains at least about 50%,60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biologicalactivity of the native polypeptide (and may even have a higher level ofactivity than the native polypeptide). A “non-functional” polypeptide isone that exhibits essentially no detectable biological activity normallyassociated with the polypeptide (e.g., at most, only an insignificantamount, e.g., less than about 10% or even 5%).

[0059] As used. herein, an “isolated” nucleic acid (e.g., an “isolatedDNA” or an “isolated vector genome”) means a nucleic acid separated orsubstantially free from at least some of the other components of thenaturally occurring organism or virus, for example, the cell or viralstructural components or other polypeptides or nucleic acids commonlyfound associated with the nucleic acid.

[0060] Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

[0061] A “therapeutically effective” amount as used herein is an amountthat is sufficient to provide some improvement or benefit to thesubject. Alternatively stated, a “therapeutically-effective” amount isan amount that will provide some alleviation, mitigation or decrease inat least one clinical symptom in the subject. To illustrate, in the caseof GAA deficiency, an amount that provides some alleviation, mitigationor decrease in at least one clinical symptom of GAA deficiency (e.g.,reduced glycogen stores in skeletal, diaphragm and/or cardiac muscle,improved muscle strength and function, improved pulmonary function,improved motor development or attainment of motor developmentalmilestones, reduction in need for or prevention of need forventilator-support, prevention of cardiac or cardiorespiratory failure,reduced premature mortality, and the like). Those skilled in the-artwill appreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

[0062] By the terms “treating” or “treatment of,” it is intended thatthe severity of the patient's condition is reduced or at least partiallyimproved and that some alleviation, mitigation, delay or decrease in atleast one clinical symptom is achieved.

[0063] A “reduction in glycogen stores” in a tissue is intended toindicate about a 25%, 35%, 40%, 50%, 60%, 75%, 85%, 90% 95% or morereduction in total glycogen in a particular tissue, unless otherwiseindicated (e.g., a reduction in Iysosomal glycogen stores or in pooledtissues).

[0064] By the term “express” or “expression” of a nucleic acid codingsequence, in particular a GAA coding sequence, it is meant that thesequence is transcribed, and optionally, translated. Generally, however,according to the present invention, the term “express” or “expression”is intended to refer to transcription and translation of the codingsequence resulting in production of the encoded polypeptide.

[0065] By “enhanced” or “enhancement” (or grammatical variations thereofwith respect to nucleic acid expression or polypeptide production, it ismeant an increase and/or prolongation of steady-state levels of theindicated nucleic acid or polypeptide, e.g., by at least about 20%, 25%,40%, 50%, 60%, 75%, 2-fold, 2.5-fold, 3-fold, 5-fold, 10-fold, 15-fold,20-fold, 30-fold, 50-fold, 100-fold or more.

[0066] Unless indicated otherwise, the terms “enhanced” or “enhancement”(or grammatical variations thereof) with respect to polypeptidesecretion indicates an increase in the relative proportion ofpolypeptide that is secreted from the cell, e.g., by at least about 20%,25%, 40%, 50%, 60%, 75%, 2-fold, 2.5-fold, 3-fold, 5-fold, 10-fold,15-fold, 20-fold, 30-foid, 50-fold, 100-fold or more.

[0067] II. Improved Constructs for Producing Lysosomal Polypeptides.

[0068] As described in more detail below, the present invention providesimproved constructs for producing lysosomal polypeptides, e.g.,polypeptides that are targeted to the, lysosomes. As known in the art,many lysosomal proteins are characterized by the presence ofmannose-6-phosphate residues, and in embodiments of the invention thelysosomal polypeptide comprises mannose-6-phosphate glycosylation. Inother representative embodiments, the lysosomal polypeptide is one thatis associated with a lysosomal-storage disease (e.g., because of adeficiency or defect in the lysosomal polypeptide). By “associated witha lysosomal storage disease”, it is meant that the lysosomal polypeptideis one that is deficient or defective in a lysosomal storage disorder,or is otherwise a causative agent in a lysosomal storage disorder.

[0069] As known in the art, there are a multitude of lysosomal storagediseases. Exemplary lysosomal storage disease include, but are notlimited to, glycogen storage disease type II (GSD II or Pompe Disease),GM1 gangliosidosis, Tay-Sachs disease, GM2 gangliosidosis (AB variant),Sandhoff disease, Fabry disease, Gaucher disease, metachromaticleukodystrophy, Krabbe disease, Niemann-Pick disease (Types A-D), Farberdisease, Wolman disease, Hurler Syndrome (MPS III), Scheie Syndrome (MPSIS), Hurler-Scheie Syndrome (MPS IH/S), Hunter Syndrome (MPS II),Sanfilippo A Syndrome (MPS IIIA), Sanfilippo B Syndrome (MPS IIIB),.Sanfilippo C Syndrome (MPS IIIC), Sanfilippo D Syndrome (MPS IIID),Morquio A disease (MPS IVA), Morquio B disease (MPS IV B),Maroteaux-Lamy disease (MPS VI), Sly Syndrome (MPS VII), α-mannosidosis,β-mannosidosis, fucosidosis, aspartylglucosaminuria, sialidosis(mucolipidosis I), mucolipidosis II (I-Cell disease), mucolipidosis III(pseudo-Hurler polydystrophy), mucolipidosis IV, galactosialidosis(Goldberg Syndrorme), Schindler disease, cystinosis, Salla disease,infantile sialic acid storage disease, Batten disease (juvenile neuronalceroid lipofuscinosis), infantile neuronal ceroid lipofuscinosis, andprosaposin.

[0070] Lysosomal polypeptides that are associated with lysosomal storagediseases according to the present invention include, but are not limitedto, lysosomal acid α-glucosidase (GAA; also known as acid maltase),α-galactosidase A, β-galactosidase, β-hexosaminidase A, β-hexosaminidaseB, GM₂ activator protein, glucocerebrosidase, arylsulfatase A,galactosylceramidase, acid sphingomyelinase, acid ceramidase, acidlipase, α-L-iduronidase, iduronate sulfatase, heparan N-sulfatase,α-N-acetylglucosaminidase, glucosaminide acetyltransferase,N-acetylglucosamine-6-sulfatase, arylsulfatase B, β-glucuronidase,α-mannbsidase, β-mannosidase, α-L-fucosidase,N-aspartyl-β-glucosaminidase, N-acetylgalactosamine 4-sulfatase,α-neuraminidase, lysosomal protective protein,α-N-acetyl-galactosaminidase, N-acetylglucosamine-1-phosphotransferase,cystine transport protein, sialic acid transport protein, the CLN3 geneproduct, palmitoyl-protein thioesterase, saposin A, saposin B, saposinC, and saposin D.

[0071] Lysosomal acid α-glucosidase or “GAA” (E.C. 3.2.1.20)(1,4-α-D-glucan glucohydrolase), is an exc-1,4-α-D-glucosidase thathydrolyses both α-1,4 and α-1,6 linkages of oligosaccharides to liberateglucose. A deficiency in GAA results in glycogen storage disease type II(GSDII), also referred to as Pompe disease (although this term formallyrefers to the infantile onset form of the disease). It catalyzes thecomplete degradation of glycogen with slowing at branching points. The28 kb human acid α-glucosidase gene on chromosome 17 encodes a 3.6kb-mRNA which produces a 951 amino acid polypeptide (Hoefsloot et al.,(1988) EMBO J. 7:1697; Martiniuk et al., (1990) DNA and Cell Biology9:85). The enzyme receives co-translational N-linked glycosylation inthe endoplasmic reticulum. It is synthesized as a 110-kDa precursorform, which matures by extensive glycosylation modification,phosphorylation and by proteolytic processing through an approximately90-kDa endosomal intermediate into the final lysosomal 76 and 67 kDaforms (Hoefsloot, (1988) EMBO J. 7:1697; Hoefsloot et al., (1990)Biochem. J. 272:485; Wisselaar et al., (1993) J. Biol. Chem. 268:2223;Hermans et al., (1993) Biochem. J. 289:681).

[0072] In patients with GSD II, a deficiency of acid α-glucosidasecauses massive accumulation of glycogen in lysosomes, disruptingcellular function (Hirschhorn, R. and Reuser, A. J. (2001), in TheMetabolic and Molecular Basis for Inherited Disease, (eds, Scriver, C.R. et al.) pages 3389-3419 (McGraw-Hill, New York). In the most commoninfantile form, patients exhibit progressive muscle degeneration andcardiomyopathy and die before two years of age. Severe debilitation ispresent in the juvenile and adult onset forms.

[0073] The term “GAA” or “GAA polypeptide,” as used herein, encompassesmature (˜76 or ˜67 kDa) and precursor (e.g., ˜110 kDa) GAA as well asmodified (e.g., truncated or mutated by insertion(s), deletion(s) and/orsubstitution(s)) GAA proteins or fragments thereof that retainbiological function (i.e., have at least one biological activity of thenative GAA protein, e.g., can hydrolyze glycogen, as defined above) andGAA variants (e.g., GAA II as described by Kunita et al., (1997)Biochemica et Biophysica Acta 1362:269; GAA polymorphisms and SNPs aredescribed by Hirschhorn, R. and Reuser, A. J. (2001) In The Metabolicand Molecular Basis for Inherited Disease (Scriver, C. R., Beaudet, A.L., Sly, W. S. & Valle, D. Eds.), pp. 3389-3419. McGraw-Hill, New York,see pages 3403-3405; each incorporated herein by reference in itsentirety). Any GAA coding sequence known in the art may be used, forexample, see the coding sequences of FIGS. 8 and 9; GenBank Accessionnumber NM_(—)00152 and Hoefsloot et al., (1988) EMBO J. 7:1697 and VanHove et al., (1996) Proc. Natl. Acad. Sci. USA 93:65 (human), GenBankAccession number NM_(—)008064 (mouse), and Kunita et al., (1997)Biochemica et Biophysica Acta 1362:269 (quail); the disclosures of whichare incorporated herein by reference for their teachings of GAA codingand noncoding sequences.

[0074] Likewise, the term “lysosomal polypeptide,” as used herein,encompasses mature and precursor lysosomal polypeptides as well asmodified (e.g., truncated or mutated by insertion(s), deletion(s) and/orsubstitution(s)) lysosomal polypeptides or fragments thereof that retainbiological function (i.e., have at least one biological activity of thenative lysosomal polypeptide) and lysosomal polypeptide variants.

[0075] The coding sequence of the lysosomal polypeptide can be derivedfrom any source, including avian and mammalian species. The term “avian”as used herein includes, but is not limited to, chickens, ducks, geese,quail, turkeys and pheasants. The term “mammal” as used herein includes,but is not limited to, humans, simians and other non-human primates,bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.In embodiments of the invention, the nucleic acids of the inventionencode a human, mouse or quail lysosomal polypeptide.

[0076] A. Constructs for Targeting Lysosomal Polypeptides to theSecretory Pathway.

[0077] Lysosomal proteins generally have amino-terminal signal peptidesthat co-translationally transfer the nascent proteins to the lumen ofthe endoplasmic reticulum. It is believed that lysosomal polypeptidesdiverge from the secretory pathway and are directed to the lysosome byat least three distinct pathways (see, e.g., Wisselaar et al., (1993) J.Biol. Chem. 268:2223-31). The best studied of these involves thepost-translational addition of mannose-6-phosphate residues that arerecognized by the phosphomannosyl receptor, which directs thetransport,of the polypeptide to the lysosome.

[0078] The first 27 amino acids of the human GAA polypeptide are typicalof signal peptides of lysosomal and secretory proteins. GAA may betargeted to lysosomes via the phosphomannosyl receptor and/or bysequences associated with the delayed cleavage of the signal peptide(Hirschhorn, R. and Reuser, A. J. (2001), in The Metabolic and MolecularBasis for Inherited Disease, (eds, Scriver, C. R. et al.) pages3389-3419 (McGraw-Hill, New York). A membrane-bound precursor form ofthe enzyme (i.e., anchored by the uncleaved signal peptide) has beenidentified in the lumen of the endoplasmic reticulum (see, e.g.,Wisselaar et al., (1993) J. Biol. Chem. 268:2223-31).

[0079] The present invention provides isolated nucleic acids encodinglysosomal polypeptides (e.g., GAA) that are fused to a signal peptidethat enhances targeting of the polypeptide to the secretory pathway.Secretion of lysosomal polypeptides from the cell provides a number ofadvantages. For example, in recombinant protein production systems (bothcultured cells/tissues or whole animals systems), it is generallypreferable to purify a secreted polypeptide from the extracellularmedium or fluids rather than harvesting the cells and isolatingintracellular protein. With respect to therapeutic methods, it has beenshown that administration of an Ad vector encoding hGAA that wastargeted to liver in a GAA knock-out mouse model reversed glycogenaccumulation in skeletal and cardiac muscle by secretion, of hGAA fromthe liver and uptake by affected tissues (Amalfitano et al., Proc. Natl.Acad. Sci. USA 96:8861-8866). Presumably, secretion of significantamounts of GAA (i.e., rather than lysosomal targeting) was a result ofover-expression of the GAA transgene delivered by the Ad vector andsaturation of the “scavenger” system that normally redirectsextracellular lysosomal proteins to the lysosome.

[0080] The present invention advantageously provides improved constructsthat enhance secretion of lysosomal polypeptides from the transduced ortransfected cell. These constructs facilitate the use of alternativedelivery systems (e.g., AAV vectors for liver delivery), enhance thesecretion of lysosomal polypeptides (e.g., for in vivo gene delivery orin vitro enzyme production), and can reduce or avoid cytotoxicity ororgan toxicity (e.g., hepatotoxicity) that may result fromover-accumulation of recombinant protein in the depot organ.

[0081] Accordingly, the invention encompasses isolated nucleic acidsencoding a chimeric polypeptide comprising a secretory signal sequenceoperably linked to a lysosomal polypeptide (e.g., GAA) as well as thechimeric polypeptides. The secretory signal sequence is foreign to(e.g., exogenous to) the lysosomal polypeptide. While those skilled inthe art will appreciate that secretory signal sequences are typically atthe amino-terminus of the nascent polypeptide, the secretory signalsequence according to the present invention can be located at anyposition within the chimeric polypeptide (e.g., N-terminal, within themature polypeptide, or C-terminal) as long as it functions as asecretory signal sequence (e.g., enhances secretion of the lysosomalpolypeptide) and does not render the lysosomal polypeptidenon-functional.

[0082] As used herein, the term “secretory signal sequence” orvariations thereof are intended to refer to amino acid sequences thatfunction to enhance (as defined above) secretion of an operably linkedlysosomal polypeptide from the cell as compared with the level ofsecretion seen with the native lysosomal polypeptide. As defined above,by “enhanced” secretion, it is meant that the relative proportion oflysosomal polypeptide synthesized by the cell that is secreted from thecell is increased; it is not necessary that the absolute amount ofsecreted protein is also increased. In particular embodiments of theinvention, essentially all (i.e., at least 95%, 97%, 98%, 99% or more)of the polypeptide is secreted. It is not necessary, however, thatessentially all or even most of the lysosomal polypeptide is secreted,as long as the level of secretion is enhanced as compared with thenative lysosomal polypeptide.

[0083] In particular embodiments, at least about 50%, 60%, 75%, 85%,90%, 95%, 98% or more of the lysosomal polypeptide is secreted from thecell.

[0084] The relative proportion of newly-synthesized lysosomalpolypeptide that is secreted from the cell can be routinely determinedby methods known in the art and as described in the Examples. Secretedproteins can be detected by directly measuring the protein itself (e.g.,by Western blot) or by protein activity assays (e.g., enzyme assays) ince11 culture medium, serum, milk, etc.

[0085] Generally, secretory signal sequences are cleaved within theendoplasmic reticulum and, in particular embodiments of the invention,the secretory signal sequence is cleaved prior to secretion. It is notnecessary, however, that the secretory signal sequence is cleaved aslong as secretion of the lysosomal polypeptide from the cell is enhancedand the lysosomal polypeptide is functional. Thus, in embodiments of theinvention, the secretory signal sequence is partially or entirelyretained.

[0086] Thus, in particular embodiments of the invention, an isolatednucleic acid encoding a chimeric polypeptide comprising a lysosomalpolypeptide operably linked to a secretory signal sequence is deliveredto a cell, and the chimeric polypeptide is produced and the lysosomalpolypeptide secreted from the cell. The lysosomal polypeptide can besecreted after cleavage of all or part of the secretory signal sequence.Alternatively, the lysosomal polypeptide can retain the secretory signalsequence (i.e., the secretory signal is not cleaved). Thus, in thiscontext, the “lysosomal polypeptide” can be a chimeric polypeptide.

[0087] Those skilled in the art will further understand that thechimeric polypeptide can contain additional amino acids, e.g., as aresult of manipulations of the nucleic acid construct such as theaddition of a restriction site, as long as these additional amino acidsdo not render the secretory signal sequence or the lysosomal polypeptidenon-functional. The additional amino acids can be cleaved or can beretained by the mature polypeptide as long as retention does not resultin a nonfunctional polypeptide.

[0088] In representative embodiments, the secretory signal peptidereplaces most, essentially all or all of the leader sequence found inthe native lysosomal polypeptide. In particular embodiments, most or allof the native leader sequence is retained, as long as secretion of thelysosomal polypeptide is enhanced and the mature lysosomal polypeptideis functional.

[0089] The secretory signal sequence can be derived in whole or in partfrom the secretory signal of a secreted polypeptide (i.e., from theprecursor) and/or can be in whole or in part synthetic. The secretorysignal sequence can be from any species of origin, including animals(e.g., avians and mammals such as humans, simians and other non-humanprimates, bovines, ovines, caprines, equines, porcines, canines,felines, rats, mice, lagomorphs), plants, yeast, bacteria, protozoa orfungi. The length of the secretory signal sequence is not critical;generally, known secretory signal sequences are from about 10-15 to50-60 amino acids in length. Further, known secretory signals fromsecreted polypeptides can be altered or modified (e.g., by substitution,deletion, truncation or insertion of amino acids) as long as theresulting secretory signal sequence functions to enhance secretion of anoperably linked lysosomal polypeptide.

[0090] The secretory signal sequences of the invention can comprise,consist essentially of or consist of a naturally occurring secretorysignal sequence or a modification thereof (as described above). Numeroussecreted proteins and sequences that direct secretion from the cell areknown in the art. Exemplary secreted proteins (and their secretorysignals) include but are not limited to: erythropoietin, coagulationFactor IX, cystatin, lactotransferrin, plasma protease Cl inhibitor,apolipoproteins (e.g., APO A, C, E), MCP-1, α-2-HS-glycoprotein,α-1-microgolubilin, complement (e.g., C1Q, C3), vitronectin,lymphotoxin-α, azurocidin, VIP, metalloproteinase inhibitor 2,glypican-1, pancreatic hormone, clusterin, hepatocyte growth factor,insulin, α-1-antichymotrypsin, growth hormone, type IV collagenase,guanylin, properdin, proenkephalin A, inhibin β (e.g., A chain),prealbumin, angiogenin, lutropin (e.g., β chain), insulin-like growthfactor binding protein 1 and 2, proactivator polypeptide, fibrinogen(e.g., β chain), gastric triacylglycerol lipase, midkine, neutrophildefensins 1, 2, and 3, α-1-antitrypsin, matrix gla-protein, α-tryptase,bile-salt-activated lipase, chymotrypsinogen B, elastin, IG lambda chainV region, platelet factor 4 variant, chromogranin A, WNT-1proto-oncogene protein, oncostatin M, β-neoendorphin-dynorphin, vonWillebrand factor, plasma serine protease inhibitor, serum amyloid Aprotein, nidogen, fibronectin, rennin, osteonectin, histatin 3,phospholipase A2, cartilage matrix protein, GAA-CSF, matrilysin,MIP-2-β, neuroendocrine protein 7B2, placental protein 11, gelsolin, IGF1 and 2, M-CSF, transcobalamin I, lactase-phlorizin hydrolase, elastase2B, pepsinogen A, MIP 1-β, prolactin, trypsinogen II, gastrin-releasingpeptide II, atrial natriuretic factor, secreted alkaline phosphatase,pancreatic α-amylase, secretogranin 1, β-casein, serotransferrin, tissuefactor pathway inhibitor, follitropin β-chain, coagulation factor XII,growth hormone-releasing factor, prostate seminal plasma protein,interleukins (e.g., 2, 3, 4, 5, 9, 11), inhibin (e.g., alpha chain),angiotensinogen, thyroglobulin, IG heavy or light chains, plasminogenactivator inhibitor-1, lysozyme C, plasminogen activator,antileukoproteinase 1, statherin, fibulin-1, isoform B, uromodulin,thyroxine-binding globulin, axonin-1, endometrial α-2 globulin,interferon (e.g., alpha, beta, gamma), β-2-microglobulin,procholecystokinin, progastricsin, prostatic acid phosphatase, bonesialoprotein II, colipase, Alzheimer's amyloid A4 protein, PDGF (e.g., Aor B chain), coagulation factor V, triacylglycerol lipase,haptoglobuin-2, corticosteroid-binding globulin, triacylglycerol lipase,prorelaxin H2, follistatin 1 and 2, platelet glycoprotein IX, GCSF,VEGF, heparin cofactor II, antithrombin-III, leukemia inhibitory factor,interstitial collagenase, pleiotrophin, small inducible cytokine A1,melanin-concentrating hormone, angiotensin-converting enzyme, pancreatictrypsin inhibitor, coagulation factor VIII, α-fetoprotein,α-lactalbumin, senogelin II, kappa casein, glucagon, thyrotropin betachain, transcobalamin II, thrombospondin 1, parathyroid hormone,vasopressin copeptin, tissue factor, motilin, MPIF-1, kininogen,neuroendocrine convertase 2, stem cell factor procollagen α1 chain,plasma kallikrein, keratinocyte growth factor, as well as any othersecreted hormone, growth factor, cytokine, enzyme, coagulation factor,milk protein, immunoglobulin chain, and the like.

[0091] In other particular embodiments, the secretory signal sequence isderived in part or in whole from a secreted polypeptide that is producedby liver cells.

[0092] The secretory signal sequence of the invention can further be inwhole or in part synthetic or artificial. Synthetic or artificialsecretory signal peptides are known in the art, see e.g., Barash et al.,“Human secretory signal peptide description by hidden Markov model andgeneration of a strong artificial signal peptide for secreted proteinexpression,” Biochem. Biophys. Res. Comm. 294:835-42 (2002); thedisclosure of which is incorporated herein in its entirety. Inparticular embodiments, the secretory signal sequence comprises,consists essentially of, or consists of the artificial secretory signal:MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 5) or variations thereof having 1, 2,3, 4, or 5 amino acid substitutions (optionally, conservative amino acidsubstitutions, conservative amino acid substitutions are known in theart).

[0093] The isolated nucleic acid encoding the chimeric polypeptide canfurther comprise an “abbreviated” 3′ UTR as described in more detailbelow.

[0094] B. “Abbreviated” GAA Constructs.

[0095] The present invention is based, in part, on the discovery thatconstructs expressing lysosomal acid α-glucosidase (GAA) that aredeleted or altered (e.g., substituted) in the 3′ untranslated region(UTR), can have advantageous properties as compared with non-deleted ornon-altered constructs. For example, the efficiency of packaging a 3′UTR deleted or otherwise “abbreviated” 3′ UTR GAA construct (asdiscussed in more detail below) into viral vectors (e.g., AAV vectors)can be improved. Further, the level of expression of the GAA mRNA and/orpolypeptide can be enhanced (e.g., as a result of a higher level oftranscription and/or translation and/or longer half-life of the mRNAtranscript and/or polypeptide) as compared with a full-length construct(e.g., SEQ ID NO:1; FIG. 8).

[0096] The present invention provides isolated nucleic acids encodingGAA, comprising a coding sequence for GAA and an “abbreviated” 3′untranslated region (UTR). The “abbreviated” 3′ UTR are altered ascompared with the native GAA 3′ UTR sequence (e.g., the 3′ UTR of SEQ IDNO:1 is from nt 3301 through 3846; see also FIG. 8). In representativeembodiments, the abbreviated 3′ UTR comprises, consists essentially ofor consists of a deleted GAA 3′ UTR. In other embodiments, theabbreviated 3′ UTR is shortened as compared with the native GAA 3′ UTRand has been altered to contain a region from a heterologous 3′ UTR,which can be a partially or wholly synthetic 3′ UTR sequence. Theseembodiments of the invention are discussed in more detail below. Theisolated nucleic acids encoding GAA and comprising an abbreviated 3′ UTRof the invention can provide for higher levels of GAA polypeptideexpression. In particular embodiments, the abbreviated constructs canalso be more efficiently packaged into viral vectors (e.g., rAAVvectors).

[0097] The abbreviated 3′ UTR of the invention encode functional 3′ UTRthat, in the presence of all other necessary regulatory elements, permitthe expression of a functional GAA polypeptide from a GAA codingsequence operably associated therewith. In illustrative embodiments, theisolated nucleic acid comprising a coding sequence for GAA and anabbreviated 3′ UTR further comprises a secretory signal sequenceoperably linked to the GAA coding sequence, as described hereinabove.

[0098] While not wishing to be held to any-particular theory of theinvention, the improved properties of “abbreviated” 3′ UTR nucleic acidsencoding GAA may be a result of their shorter total size and/or removalof an inhibitory region, e.g., a region that reduces transcription,destabilizes the mRNA transcript and/or inhibits translation. Examplesof small sequences that destabilize mRNA for cytokines (see, e.g., Shawand Kamen, (1986) Cell 46:659-67; Reeves et al., (1987) Proc. Natl.Acad. Sci. USA 84:6531-35) and the HIV gag gene (see, e.g., Schwartz etal., (1992) J. Virology 66:150-59; Schwartz et al., (1992) J. Virol.66:7176-82) have been described.

[0099] In particular embodiments, the isolated nucleic acid comprisingthe GAA coding sequence and abbreviated 3′ UTR is less than about 4.5,4.4, 4.3, 4.2, 4.1, 4, 3.9 or 3.8 kb in length. To illustrate, accordingto representative embodiments, a GAA expression construct including 5′and 3′ UTR sequences is less than about 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9or 3.8 kb in length.

[0100] The isolated nucleic acid encoding GAA can further comprise a 5′UTR, which can further include all or a portion of the 5′ UTR of a GAAgene. Human GAA sequences with deletions in the 5′ UTR have beendescribed, see, e.g., Van Hove et al. (1996) Proc. Natl. Acad; Sci. USA93:65, in which nt 1-409 of the 5′ UTR of SEQ ID NO:1 (FIG. 8) have beendeleted (see also SEQ ID NO:3; FIG. 9). Alternatively, the 5′ UTR can bederived in whole or in part from a heterologous gene (i.e., a gene otherthan a GAA gene) and/or can comprise in whole or in part syntheticsequences.

[0101] As one aspect, the present invention provides isolated nucleicacids that encode GAA, where the isolated nucleic acid comprises (i) aGAA coding sequence encoding a GAA and (ii) a GAA 3′ UTR region having adeletion therein.

[0102] A “coding region encoding a GAA polypeptide” comprises nucleotidesequences that can be transcribed and translated to yield a functionalGAA polypeptide or functional fragment thereof (see above). Such codingsequences may include non-translated sequences (e.g., intron sequences).

[0103] A “GAA 3′-UTR region” refers to the non-translated nucleic acidsequences of a GAA gene that are located downstream of (i.e., 3′ to) theregions of the gene that encode the GAA protein.

[0104] By “deleted” GAA 3′ UTR, it is intended that there is an omissionof at least one nucleotide from the 3′ UTR region of the GAA expressionconstruct. Deletions can be greater than about 25, 50, 100,150, 200,300, 400 consecutive nucleotides, or more. In particular embodiments,essentially all of the 3′ UTR is deleted. By “essentially all” it ismeant that only an insignificant fragment of the intact 3′ UTR remains(i.e., less than 10, 20 or 30 nucleotides). For example, essentiallyall, but not necessarily all, of the 3′ UTR can be conveniently removedusing restriction enzymes (i.e., there may be some residual nucleotidesleft after restriction enzyme cleavage) or some untranslated nucleotidesmay remain 3′ of the coding sequence as an artifact of cloningprocedures.

[0105] Alternatively stated, at least 25%, 50%, 60%, 70%, 80%, 90%, 95%,99% or more of the GAA 3′ UTR can be deleted. As still a furtheralternative, in embodiments of the invention, the “deleted” GAA 3′ UTRis less than about 300, 250, 200, 175,150, 125,100, 75, 50, 30, 20 or 10nucleotides in length or less.

[0106] In particular embodiments of the invention, the deleted 3′ UTRcomprises, consists essentially of or consists of a deleted form of the3′ UTR in the human GAA sequence provided in SEQ ID NO:1 (i.e., the 3′UTR of SEQ ID NO.1 is from nt 3301 through 3846; see also FIG. 8). Forexample, the deleted 3′ UTR can comprise, consist essentially of orconsist of the 3′ UTR shown in SEQ ID NO:3 (i.e., the 3′ UTR of SEQ IDNO:3 is from nt 2878 through 3012; see also, FIG. 9).

[0107] Referring to the 3′ UTR in the GAA sequence of SEQ ID NO:1 andFIG. 8 (nt 3301 through 3846), the deleted 3′ UTR can comprise adeletion from nt 3301 through 3846 of SEQ ID NO:1. The deletion can alsoencompass from about nt 3400 through nt 3500, nt 3500 through nt 3600,nt 3600 through nt 3700, nt 3700 through nt 3800, nt 3800 throughnt3846, nt 3301 through nt 3450, nt 3450 through nt 3600, nt 3600through nt 3750, nt 3750 through nt 3846, nt 3301 through nt 3500, nt3400 through nt 3600, nt 3500 through nt 3700, nt 3600 through nt 3800,nt 3700 through nt 3846, nt 3301 through nt 3600, nt 3400 through nt3700, nt 3500 through nt 3800, nt 3600 through nt 3846, nt 3301 throughnt 3700, nt 3400 through nt 3800, nt 3500 through nt 3845, nt 3300through nt 3800, or nt 3400 through nt 3486.

[0108] Deletions can be intermittent, i.e., more than one region of thenucleotide sequence can be deleted to impart the functional improvementsdescribed herein. Alternatively, consecutive nucleotides can be deleted.Further, the deletion can be internal or start at either end of the 3′UTR. For example, the 3′ UTR sequence can be truncated from the 5′ or 3′end of the 3′ UTR by 50, 100, 200, 300 or 400 nt or more.

[0109] Those skilled in the art will readily appreciate that the deletedand intact (i.e., from which the deleted 3′ UTR are derived) GAA 3′ UTRregions within the scope of the present invention can deviate from thosespecifically disclosed herein and that any suitable GAA coding sequenceor GAA 3′ UTR may be employed. The GAA coding sequences and 3′ UTR cancontain other alterations such as substitutions or insertions therein.For example, It will be understood that the GAA 3′ UTR can contain someheterologous sequence(s) (e.g., the polyA signal may be from anothergene, such as the human or bovine growth hormone gene).

[0110] In embodiments of the invention, the 3′ UTR deleted nucleic acidencoding GAA will hybridize to the 3′ UTR deleted nucleic acid sequencesspecifically disclosed herein (i.e., SEQ ID NO:3) under standardconditions as known by those skilled in the art and encode a functionalGAA polypeptide (as defined above).

[0111] In other embodiments, the deleted GAA 3′ UTR of the inventionwill hybridize to the deleted GAA 3′ UTR sequences specificallydisclosed herein (e.g., nt 2878 to 3012 of SEQ ID NO:3) under standardconditions as known by those skilled in the art and permit theexpression of a functional GAA polypeptide from a GAA coding sequenceoperably associated therewith.

[0112] In still further embodiments, the coding sequence of the isolatednucleic acid encoding GAA will hybridize to the sequences encoding GAAspecifically disclosed herein (e.g., nt 442 to nt 3300 of SEQ ID NO:1)under standard conditions as known by those skilled in the art andencode a functional GAA polypeptide.

[0113] For example, hybridization of such sequences can be carried outunder conditions of reduced stringency, medium stringency or evenstringent conditions (e.g., conditions represented by a wash stringencyof 35-40% formamide with 5× Denhardt's solution, 0.5% SDS and 1×SSPE at37° C.; conditions represented by a wash stringency of 40-45% formamidewith 5× Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; andconditions represented by a wash stringency of 50% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively) to thesequences specifically disclosed herein. See, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring HarborLaboratory).

[0114] Alternatively stated, in embodiments of the inventions 3′ UTRdeleted nucleic acid encoding GAA of the invention have at least about60%, 70%, 80%, 90%, 95%, 97%, 98% or higher nucleotide sequence homologywith the isolated nucleic acid sequences specifically disclosed herein(or fragments thereof) -and encode a functional GAA protein (mature orprecursor forms).

[0115] Likewise, in embodiments of the invention, the deleted 3′ UTRaccording to the present invention have at least about 60%, 70%, 80%,90%, 95%, 97%, 98%, or higher nucleotide sequence homology with theisolated nucleic acid sequences'specifically disclosed herein (orfragments thereof) and permit the expression of a functional GAApolypeptide from a GAA coding sequence operably associated therewith.

[0116] Further, in embodiments of the invention, the coding region ofthe isolated nucleic acids encoding GAA of the invention have at leastabout 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher nucleotide sequencehomology with the isolated nucleic acid sequences specifically disclosedherein (or fragments thereof) and encode a functional GAA polypeptide.

[0117] It will be appreciated by those skilled in the art that there maybe variability in the polynucleotides that encode the GAA proteins ofthe present invention due to the degeneracy of the genetic code. Thedegeneracy of the genetic code, which allows different nucleic acidsequences to code for the same polypeptide, is well known in theliterature (see Table 1). TABLE 1 Amino Acids Codons Alanine Ala A GCAGCC GCG GCT Cysteine Cys C TGC TGT Aspartic acid Asp D GAC GAT Glutamicacid Glu E GAA GAG Phenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGGGGT Histidine His H CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys KAAA AAG Leucine Leu L TTA TTG CTA CTC CTG CTT Methionine Met M ATGAsparagine Asn N AAC AAT Proline Pro P CCA CCC CCG CCT Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGT Serine Ser S AGC ACT TCATCC TCG TCT Threonine Thr T ACA AGC ACG ACT Valine Val V GTA GTC GTG GTTTryptophan Trp W TGG Tyrosine Tyr Y TAC TAT

[0118] Further, in other embodiments, the isolated nucleic acids of theinvention encompass those nucleic acids encoding GAA polypeptides thathave at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or higheramino acid sequence homology with the polypeptide sequences specificallydisclosed herein (or fragments thereof) and encode a functional GAApolypeptide.

[0119] As is known in the art, a number of different programs can beused to identify whether a nucleic acid or polypeptide has sequenceidentity or similarity to a known sequence. Sequence identity and/orsimilarity can be determined using standard techniques known in the art,including, but not limited to, the local sequence identity algorithm ofSmith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequenceidentity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85,2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer-Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12, 387-395 (1984), preferably using the defaultsettings, or by inspection.

[0120] An example of a useful algorithm is PILEUP, which creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments. It can also plot a tree showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35, 351-360 (1987); the method is similar to thatdescribed by Higgins & Sharp, CABIOS 5, 151-153 (1989).

[0121] Another example of a useful algorithm is the BLAST algorithm,described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) andKarlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). Aparticularly useful BLAST program is the WU-BLAST-2 program which wasobtained from Altschul et al., Methods in Enzymology, 266, 460-480(1996); http://blast.wustI/edu/blast/README.html. WU-BLAST-2 usesseveral search parameters, which are preferably set to the defaultvalues. The parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

[0122] An additional useful algorithm is gapped BLAST as reported byAltschul et al. Nucleic Acids Res. 25, 3389-3402.

[0123] A percentage amino acid sequence identity value can be determinedby the number of matching identical residues divided by the total numberof residues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

[0124] The alignment can include the introduction of gaps in thesequences to be aligned. In addition, for sequences which contain eithermore or fewer amino acids than the polypeptides specifically disclosedherein, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. Thus, forexample, sequence identity of sequences shorter than a sequencespecifically disclosed herein, will be determined using the number ofamino acids in the shorter sequence, in one embodiment. In percentidentity calculations relative weight is not assigned to variousmanifestations of sequence variation, such as, insertions, deletions,substitutions, etc.

[0125] In one embodiment, only identities are scored positively (+1) andall forms of sequence variation including gaps are assigned a value of“0,” which obviates the need for a weighted scale or parameters asdescribed below for sequence similarity calculations. Percent sequenceidentity can be calculated, for example, by dividing the number ofmatching identical residues by the total number of residues of the“shorter” sequence in the aligned region and multiplying by 100. The“longer” sequence is the one having the most actual residues in thealigned region.

[0126] In a further representative embodiment of the invention, theisolated nucleic acid encoding GAA comprises an abbreviated 3′ UTR thatis shorter than the 3′ UTR found in the native gene (e.g., nt 3301 to nt3846 of SEQ ID NO:1), to illustrate, is less than about 75%, 50%, 40%,30%, 200o, 10%, 5%, 4%, or less, of the size of the 3′ UTR found in thenative GAA gene, and comprises a heterologous region that is substitutedfor all or a portion of the native 3′ UTR. According to this embodiment,all or at least a portion of the 3′ UTR is heterologous to the GAAcoding region (i.e:, is not derived from the 3′ UTR of a GAA gene). Theheterologous segment can include all or a portion of a 3′ UTR of anothergene and/or can be partially or completely synthetic. In particularembodiments, the heterologous region can be about 300, 250, 200, 175,125, 100, 75, 50, 30, 20 or 10 nucleotides in length or less. Toillustrate, the substituted 3′ UTR can include all or a portion of thebovine or human growth hormone 3′ UTR.

[0127] According to some embodiments of the invention, the total size ofthe abbreviated 3′ UTR is less than about 300, 250, 200, 175, 150, 125,100, 75, 50, 30, 20.or 10 nucleotides.

[0128] By “substitute,” “substituted” or “substitution” in reference tothe 3′ UTR is meant that a portion of the naturally-occurring nucleotidesequence of the GAA 3′ UTR has been replaced by a heterologousnucleotide sequence, resulting in a nucleic acid encoding GAA having theadvantages described herein.

[0129] According to the present invention, an “abbreviated” 3′ UTRencompasses both deleted GAA 3′ UTR (described at length above) and thesubstituted/shortened 3′ UTR described in the preceding paragraphs. Theabbreviated 3′ UTR of the invention may be DNA or RNA, or a chimerathereof.

[0130] It still other embodiments of the invention, the “abbreviated” 3′UTR is less than about 300, 250, 200,150 or 100 nucleotides in lengthand is derived in whole or in part from a native GAA 3′ UTR and/or inwhole or in part from a heterologous 3′ UTR. Further, the heterologous3′ UTR sequences can be derived from another gene or can be in whole orin part a synthetic sequence.

[0131] As described in more detail below, the abbreviated 3′ UTR nucleicacids of the invention can, upon introduction into a target cell (e.g.,a liver cell), express GAA polypeptide at an enhanced (as defined above)level as compared with a cell expressing GAA polypeptide from acomparable construct that contains a full-length GAA 3′ UTR (e.g., SEQID NO:1).

[0132] III. Nucleic Acid Delivery Vectors.

[0133] The methods of the present invention provide a means fordelivering and, optionally, expressing lysosomal polypeptides such asGAA in a broad range of host cells, including both dividing andnon-dividing cells in vitro or in vivo. In embodiments of the invention,the nucleic acid may be stably introduced into the target cell, forexample, by integration into the genome of the cell or by persistentexpression from stably maintained episomes (e.g., derived from EpsteinBarr Virus). Alternatively, the isolated nucleic acid can be transientlyexpressed in the cell.

[0134] The isolated nucleic acids, vectors, cells, methods andpharmaceutical formulations of the present invention are additionallyuseful in a method of administering lysosomal polypeptides such as GAAto a subject in need thereof. In this manner, the polypeptide can thusbe produced in vivo in the subject. The subject may have a deficiency ofthe polypeptide, or the production of a foreign polypeptide in thesubject may impart some therapeutic effect. Pharmaceutical formulationsand methods of delivering lysosomal polypeptides such as GAA fortherapeutic purposes are described in more detail in Section V below.

[0135] Alternatively, a polynucleotide encoding and expressing thelysosomal polypeptide (e.g., GAA) can be administered to a subject sothat the polypeptide is expressed by the subject and purified therefrom,i.e., as a source of recombinant polypeptide. According to thisembodiment, it is preferred that the polypeptide is secreted into thesystemic circulation or into another body fluid (e.g., milk, lymph,spinal fluid, urine) that is easily collected and from which thepolypeptide can be further purified. Alternatively, the polypeptide canbe expressed in avian species and deposited in, and convenientlyisolated from, egg proteins.

[0136] Likewise, the polypeptide can be expressed transiently or stablyin a cell culture system. In particular embodiments, the polypeptide issecreted into the medium and can be purified therefrom using routinetechniques known in the art. Additionally, or alternatively, the cellscan be lysed and the recombinant polypeptide can be purified from thecell lysate. The cell may be a bacterial, protozoan, plant, yeast,fungus, or animal cell. The cell can be an animal cell (e.g., insect,avian or mammalian). Representative mammalian cells include but are notlimited to fibroblasts, CHO cells, 293 cells, HT1080 cells, HeLa cellsand C10 cells.

[0137] In the case of GAA, the recombinant GAA polypeptide can beisolated using standard techniques and administered to subjects with GAAdeficiency using enzyme replacement protocols (see, e.g., Van der Ploeget al., (1991) J. Clin. Invest 87:513).

[0138] Transfer of a nucleic acid encoding a lysosomal polypeptide(e.g., GAA) to a cell in culture or to a subject also finds use as amodel for understanding disease states such as GSD II and forinvestigating the biology of these polypeptides.

[0139] Still further, the instant invention finds use in screeningmethods, whereby the polypeptide is transiently or stably expressed in acell culture system or animal model and used as a target for drugdiscovery.

[0140] Methods of producing lysosomal polypeptides such as GAA incultured cells or organisms for the purposes described above are setforth in more detail in Section IV below.

[0141] It will be apparent to those skilled in the art that any suitablevector may be used to deliver the isolated nucleic acids of theinvention to the target cell(s) or subject of interest. The choice ofdelivery vector may be made based on a number of factors known in theart, including age and species of the target host, in vitro vs. in vivodelivery, level and persistence of expression desired, intended purpose(e.g., for therapy or enzyme production), the target cell or organ,route of delivery, size of the isolated nucleic acid, safety concerns,and the like.

[0142] Any suitable vector known in the art can be used to deliver, andoptionally, express the isolated nucleic acids of the invention,including, virus vectors (e.g., retrovirus, adenovirus, adeno-associatedvirus, or herpes simplex virus), lipid vectors, poly-lysine vectors,synthetic polyamino polymer vectors that are used with nucleic acidmolecules, such as a plasmid, and the like.

[0143] Any viral vector that is known in the art may be used in thepresent invention. Examples of such viral vectors include, but are notlimited to vectors derived from: Adenoviridae; Birnaviridae;Bunyaviridae; Caliciviridae, Capillovirus group; Carlavirus group;Carmovirus virus group; Group Caulimovirus; Closterovirus Group;Commelina yellow mottle virus group; Comovirus virus group;Coronaviridae; PM2 phage group; Corcicoviridae; Group Cryptic virus;group Cryptovirus; Cucumovirus virus group family ([PHgr]6 phage group;Cysioviridae; Group Carnation ringspot; Dianthovirus virus group; GroupBroad bean wilt; Fabavirus virus group; Filoviridae; Flaviviridae;Furovirus group; Group Germinivirus; Group Giardiavirus; Hepadnaviridae;Herpesviridae; Hordeivirus virus group; Illarvirus virus group;Inoviridae; Iridoviridae; Leviviridae; Lipothrixviridae; Luteovirusgroup; Marafivirus virus group; Maize chlorotic dwarf virus group;icroviridae; Myoviridae; Necrovirus group; Nepovirus virus group;Nodaviridae; Orthomyxoviridae; Papovaviridae; Paramyxoviridae; Parsnipyellow fleck virus group; Partitiviridae; Parvoviridae; Pea enationmosaic virus group; Phycodnaviridae; Picomaviridae; Plasmaviridae;Prodoviridae; Polydnaviridae; Potexvirus group; Potyvirus; Poxviridae;Reoviridae; Retroviridae; Rhabdoviridae; Group Rhizidiovirus;Siphoviridae; Sobemovirus group; SSV 1-Type Phages; Tectiviridae;Tenuivirus; Tetraviridae; Group Tobamovirus; Group Tobravirus;Togaviridae; Group Tombusvirus; Group Torovirus; Totiviridae; GroupTymovirus; and plant virus satellites.

[0144] Protocols for producing recombinant viral vectors and for usingviral vectors for nucleic acid delivery can be found in CurrentProtocols in Molecular Biology, Ausubel, F. M. et al. (eds.) GreenePublishing Associates, (1989) and other standard laboratory manuals(e.g., Vectors for Gene Therapy. In: Current Protocols in HumanGenetics. John Wiley and Sons, Inc.: 1997).

[0145] Particularly preferred viral vectors are those previouslyemployed for the delivery of transgenes including, for example,retrovirus, adenovirus, AAV, herpes virus, hybrid adenovirus-AAV, andpoxvirus vectors. In particular embodiments, the vector is an adenovirusvector, AAV vector or hybrid Ad-AAV vector.

[0146] In certain preferred embodiments of the present invention, thedelivery vector is an adenovirus vector. The term “adenovirus” as usedherein is intended to encompass all adenoviruses, including theMastadenovirus and Aviadenovirus genera. To date, at least forty-sevenhuman serotypes of adenoviruses have been identified (see, e.g., FIELDSet al., VIROLOGY, volume 2, chapter 67 (3d ed., Lippincott-RavenPublishers)). Preferably, the adenovirus is a serogroup C adenovirus,still more preferably the adenovirus is serotype 2 (Ad2) or serotype 5(Ad5).

[0147] The various regions of the adenovirus genome have been mapped andare understood by those skilled in the art (see, e.g., FIELDS et al.,VIROLOGY, volume 2, chapters 67 and 68 (3d ed., Lippincott-RavenPublishers)). The genomic sequences of the various Ad serotypes, as wellas the nucleotide sequence of the particular coding regions of the Adgenome, are known in the art and may be accessed, e.g., from GenBank andNCBI (see, e.g., GenBank Accession Nos. J0917; M73260, X73487, AF108105,L19443, NC 003266 and NCBI Accession Nos. NC 001405, NC 001460, NC002067, NC 00454).

[0148] Those skilled in the art will appreciate that the inventiveadenovirus vectors may be modified or “targeted” as described in Douglaset al., (i996) Nature Biotechnology 14:1574; U.S. Pat. No. 5,922,315 toRoy et al.; U.S. Pat. No. 5,770,442 to Wickham et al.; and/or U.S. Pat.No.5,71-2,136 to Wickham et al.

[0149] An adenovirus vector genome or rAd vector genome will typicallycomprise the Ad terminal repeat sequences and packaging signal. An“adenovirus particle” or “recombinant adenovirus particle” comprises anadenovirus vector genome or recombinant adenovirus vector genome,respectively, packaged within an adenovirus capsid. Generally, theadenovirus vector genome is most stable at sizes of about 28 kb to 38 kb(approximately 75% to 105% of the native genome size). In the case of anadenovirus vector containing large deletions and a relatively smalltransgene, “stuffer DNA” can be used to maintain the total size of thevector within the desired range by methods known in the art.

[0150] Normally adenoviruses bind to a cell surface receptor (CAR) ofsusceptible cells via the knob domain of the fiber protein on the virussurface. The fiber knob receptor is a 45 kDa cell surface protein whichhas potential sites for both glycosylation and phosphorylation.(Bergelson, et al., (1997) Science 275:1320-1323. A secondary method ofentry for adenovirus is through integrins present on the cell surface.Arginine-Glycine-Aspartic Acid (RGD) sequences of the adenoviral pentonbase protein bind integrins on the cell'surface.

[0151] The genome of an adenovirus can be manipulated such that itencodes and expresses a gene product of interest but is inactivated interms of its ability to replicate in a normal lytic viral life cycle.See, for example, Berkner et al. (1988) Bio Techniques 6:616; Rosenfeldet al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1 324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7, etc.) are known to those skilled in the art. Recombinantadenoviruses can be advantageous in certain circumstances in that theyare not capable of infecting nondividing cells and can be used to infecta wide variety of cell types, including epithelial cells. Furthermore,the virus particle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situations whereintroduced DNA becomes integrated into the host genome (e.g., retroviralDNA). Moreover, the carrying capacity of the adenoviral genome forforeign DNA is large relative to other nucleic acid delivery vectors(Haj-Ahmand and Graham (1986) J. Virol. 57:267).

[0152] In particular embodiments, the adenovirus genome contains adeletion therein, so that at least one of the adenovirus gene regionsdoes not encode a functional protein. For example, first-generationadenovirus vectors are typically deleted for the E1 genes and packagedusing a cell that expresses the E1 proteins (e.g., 293 cells). The E3region is also frequently deleted as well, as there is no need forcomplementation of this deletion. In addition, deletions in the E4, E2a,protein IX, and fiber protein regions have been described, e.g., byArmentano et al, (1997) J. Virology 71:2408, Gao et al., (1996) J.Virology 70:8934, Dedieu et al., (1997) J. Virology 71;4626, Wang etal., (1997) Gene Therapy 4:393, and U.S. Pat. No. 5,882,877 to Gregoryet al. (the disclosures of which are incorporated herein in theirentirety). Preferably, the deletions are selected to avoid toxicity tothe packaging cell. Wang et al., (1997) Gene Therapy 4:393, hasdescribed toxicity from constitutive co-expression of the E4 and E1genes by a packaging cell line. Toxicity may be avoided by regulatingexpression of the E1 and/or E4 gene products by an inducible, ratherthan a constitutive, promoter. Combinations of deletions that avoidtoxicity or other deleterious effects on the host cell can be routinelyselected by those skilled in the art.

[0153] As further examples, in particular embodiments, the adenovirus isdeleted in the polymerase (pol), preterminal protein (pTP), IVa2 and/or100K regions (see, e.g., U.S. Pat. No. 6,328,958; PCT publication WO00/12740;, and PCT publication WO 02/098466; Ding et al., (2002) Mol.Ther. 5:436; Hodges et al., J. Virol. 75:5913; Ding et al., (2001) HumGene Ther 12:955; the disclosures of which are incorporated herein byreference in their entireties for the teachings of how to make and usedeleted adenovirus vectors for nucleic acid delivery). In representativeembodiments, the vector is a [E1-, E3-, pol-]Ad, [E1-, E3-, pTP-]Ad,[E1-, E3-,pol1, pTP-]Ad, [E1+, 100K-]Ad or [E1a+, E1b−, 100K]Ad.

[0154] The term “deleted” adenovirus as used herein refers to theomission of at least one nucleotide from the indicated region of theadenovirus genome. Deletions can be greater than about 1, 2, 3, 5, 10,20, 50, 100, 200, or even 500 nucleotides. Deletions in the variousregions of the adenovirus genome may be about at least 1%, 5%, 10%, 25%,50%, 75%, 90%, 95%, 99%, or more of the indicated region. Alternately,the entire region of the adenovirus genome is deleted. Preferably, thedeletion will prevent or essentially prevent the expression of afunctional protein from that region. For example, it is preferred thatthe deletion in the 100K region results in the loss of expression of afunctional 100K protein from that region. In other words, even if thereis transcription across the deleted 100K region and translation of theresulting RNA transcripts, the resulting protein will be essentiallynon-functional, more preferably, completely non-functional.Alternatively, an insignificant amount of a functional protein isexpressed. In general, larger deletions are preferred as these have theadditional advantage that they will increase the carrying capacity ofthe deleted adenovirus for a heterologous nucleotide sequence ofinterest. The various regions of the adenovirus genome have been mappedand are understood by those skilled in the art (see, e.g., FIELDS etal., VIROLOGY, volume 2, chapters 67 and 68 (3d ed., Lippincott-RavenPublishers)).

[0155] Those skilled in the art will appreciate that typically, with theexception of the E3 genes, any deletions will need to be complemented inorder to propagate (replicate and package) additional virus, e.g., bytranscomplementation with a packaging cell.

[0156] In particular embodiments, the present invention excludes “guttedadenovirus” vectors (as that term is understood in the art, see e.g.,Lieber, et al., (1996) J. Virol. 70:8944-60) in which essentially all ofthe adenovirus genomic sequences are deleted. In alternate embodiments,such gutted adenovirus vectors may be an aspect of the invention.

[0157] Adeno-associated viruses (AAV) have also been employed as nucleicacid delivery vectors. For a review, see Muzyczka et al. Curr. Topics inMicro. and Immunol. (1992) 158:97-129). AAV are parvoviruses and havesmall icosahedral virions, 18-26 nanometers in diameter and contain asingle stranded DNA molecule 4-5 kilobases in size. The viruses containeither the sense or antisense strand of the DNA molecule and eitherstrand is incorporated into the virion. Two open reading frames encode aseries of Rep and Cap polypeptides. Rep polypeptides (Rep50, Rep52,Rep68 and Rep78) are involved in replication, rescue and integration ofthe AAV genome, although significant activity may be observed in theabsence of all four Rep polypeptides. The Cap proteins (VP1, VP2, VP3)form the virion capsid. Flanking the rep and cap open reading frames atthe 5′ and 3′ ends of the genome are 145 basepair inverted terminalrepeats (ITRs), the first 125 basepairs of which are capable of formingY- or T-shaped duplex structures. It has been shown that the ITRsrepresent the minimal cis sequences required for replication, rescue,packaging and integration of the AAV genome. Typically, in recormbinantAAV vectors (rAAV), the entire rep and cap coding regions are excisedand replaced with a transgene of interest.

[0158] AAV are among the few viruses that may integrate their DNA intonon-dividing cells, and exhibit a high frequency of stable integrationinto human chromosome 19 (see, for example, Flotte et al. (1992) Am. J.Respir. Cell. Mol. Biol. 7:349-356; Samulski et al., (1989) J Virol.63:3822-3828; and McLaughlin et al., (1989) J. Virol. 62:1963-1973). Avariety of nucleic acids have been introduced into different cell typesusing AAV vectors (see, for example, Hermonat et al., (1984) Proc. Natl.Acad. Sci. USA 81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol.4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39;Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte et al., (1993)J. Biol. Chem. 268:3781-3790).

[0159] A rAAV vector genome will typically comprise the AAV terminalrepeat sequences and packaging signal. An “AAV particle” or “rAAVparticle” comprises an AAV vector genome or rAAV vector genome,respectively, packaged within an AAV capsid. The rAAV vector itself neednot contain AAV genes encoding the capsid and Rep proteins. Inparticular embodiments of the invention, the rep and/or cap genes aredeleted from the AAV genome. In a representative embodiment, the rAAVvector retains only the terminal AAV sequences (ITRs) necessary forintegration, excision, replication.

[0160] Sources for the AAV capsid genes may include serotypes AAV-1,AAV-2, AAV-3 (including 3a and 3b), AAV-4, AAV-5, AAV-6, AAV-7, AAV-8,as well as bovine AAV and avian AAV, and any other virus classified bythe International Committee on Taxonomy of Viruses (ICTV) as an AAV(see, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69(4th ed., Lippincott-Raven Publishers)).

[0161] In particular embodiments, the AAV capsid genes are derived fromAAV serotypes 1, 2, 5, 6 or 8.

[0162] Because of packaging limitations, the total size of the rAAVgenome will preferably be less than about 5.2, 5, 4.8, 4.6 or 4.5 kb insize.

[0163] Any suitable method known in the art may be used to produce AAVvectors (see, e.g., U.S. Pat. No. 5,139,941; U.S. Pat. No. 5,858,775;U.S. Pat. No. 6,146,874 for illustrative methods). In one particularmethod, AAV stocks may be produced by co-transfection of a rep/capvector encoding AAV packaging functions and the template encoding theAAV vDNA into human cells infected with the helper adenovirus (Samulskiet al., (1989) J. Virology 63:3822).

[0164] In other particular embodiments, the adenovirus helper virus is ahybrid helper virus that encodes AAV Rep and/or capsid proteins. Hybridhelper Ad/AAV vectors expressing AAV rep and/or cap genes and methods ofproducing AAV stocks using these reagents are known in the art (see,e.g., U.S. Pat. No. 5,589,377; and U.S. Pat. No. 5,871,982, U.S. Pat.No. 6,251,677; and U.S. Pat. No. 6,387,368). Preferably, the hybrid Adof the invention expresses the AAV capsid proteins (i.e., VP1, VP2, andVP3). Alternatively, or additionally, the hybrid adenovirus may expressone or more of AAV Rep proteins (i.e., Rep40, Rep52, Rep68 and/orRep78). The AAV sequences may be operatively associated with atissue-specific or inducible promoter.

[0165] The AAV rep and/or cap genes may alternatively be provided by apackaging cell that stably expresses the genes (see, e.g., Gao et al.,(1998) Human Gene Therapy 9:2353; Inoue et al., (1998) J. Virol.72:7024; U.S. Pat. No. 5,837,484; WO 98/27207; U.S. Pat. No. 5,658,785;WO 96/17947).

[0166] In a representative embodiment, the present invention provides amethod of producing a rAAV particle comprising an isolated nucleic acidaccording to the invention, comprising providing to a cell: (a) anucleic acid encoding a rAAV genome comprising (i) 5′ and/or 3′ AAV ITRsequences, (ii) an isolated nucleic acid as described above (e.g., anucleic acid encoding GAA and comprising an abbreviated 3′ UTR or anucleic acid encoding a chimeric lysosomal polypeptide comprising asecretory signal sequence), and (iii) an AAV packaging signal; (b) AAVrep coding sequences sufficient for replication of the recombinant AAVgenome; (c) AAV cap coding sequences sufficient to produce a functionalAAV capsid; wherein (a) to (c) are provided to the cell under conditionssufficient for replication and packaging of the rAAV genome into the AAVcapsid, whereby AAV particles comprising the AAV capsid packaging therAAV genome are produced in the cell. Typically, the adenovirus or HSVhelper functions for AAV replication and packaging are also provided.The method may further include the step of collecting the rAAVparticles.

[0167] In still further embodiments, the delivery vector is a hybridAd-AAV delivery vector, for example, as described in the workingExamples and in U.S. Provisional Application 60/376,397 (incorporated byreference herein in its entirety for its teaching of how to make and usehybrid Ad-AAV delivery vectors). Briefly, the hybrid Ad-AAV vectorcomprises an adenovirus vector genome comprising adenovirus (i) 5′ and3′ cis-elements for viral replication and encapsidation and, further,(ii) a recombinant AAV vector genome comprising the AAV 5′ and 3′inverted terminal repeats (ITRs), an AAV packaging sequence, and aheterologous sequence(s) flanked by the AAV ITRs, where the recombinantAAV vector genome is flanked by the adenovirus 5′ and 3′ cis-elements.The adenovirus vector genome may further be deleted, as described above.

[0168] Another vector for use in the present invention comprises HerpesSimplex Virus (HSV). Herpes simplex virions have an overall diameter of150 to 200 nm and a genome consisting of one double-stranded DNAmolecule that is 120 to 200 kilobases in length. Glycoprotein D (gD) isa structural component of the HSV envelope that mediates virus entryinto host cells. The initial interaction of HSV with cell surfaceheparin sulfate proteoglycans is mediated by another glycoprotein,glycoprotein C (gC) and/or glycoprotein B (gB). This is followed byinteraction with one or more of the viral glycoproteins with cellularreceptors. Recently it has been shown that glycoprotein D of HSV bindsdirectly to Herpes virus entry mediator (HVEM) of host cells. HVEM is amember of the tumor necrosis factor receptor superfamily (Whitbeck, J.C. et al., 1997, J. Virol.; 71:6083-6093). Finally, gD, gB and thecomplex of gH and gL act individually or in combination to triggerpH-independent fusion of the viral envelope with the host cell plasmamembrane. The virus itself is transmitted by direct contact andreplicates in the skin or mucosal membranes before infecting cells ofthe nervous system for which HSV has particular tropism. It exhibitsboth a lytic and a latent function. The lytic cycle results in viralreplication and cell death. The latent function allows for the virus tobe maintained in the host for an extremely long period of time.

[0169] HSV can be modified for the delivery of transgenes to cells byproducing a vector that exhibits only the latent function forlong-term-gene maintenance. HSV vectors are useful for nucleic aciddelivery because they allow for a large DNA insert of up to or greaterthan 20 kilobases; they can be produced with extremely high titers; andthey have been shown to express transgenes for a long period of time inthe central nervous system as long as the lytic cycle does not occur.

[0170] In other preferred embodiments of the present invention, thedelivery vector of interest is a retrovirus. Retroviruses normally bindto a species specific cell surface receptor, e.g., CD4 (for HIV); CAT(for MLV-E; ecotropic Murine leukemic virus E); RAM1/GLVR2 (for murineleukemic virus-A; MLV-A); GLVR1 (for Gibbon Ape leukemia virus (GALV)and Feline leukemia virus B (FeLV-B)). The development of specializedcell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview, see Miller, (1990) Blood 76:271). A replication-defectiveretrovirus can be packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.

[0171] Yet another suitable vector is a poxvirus vector. These virusesare very complex, containing more than 100 proteins, although thedetailed structure of the virus is presently unknown. Extracellularforms of the virus have two membranes while intracellular particles onlyhave an inner membrane. The outer surface of the virus is made up oflipids and proteins that surround the biconcave core. Poxviruses arevery complex antigenically, inducing both specific and cross-reactingantibodies after infection. Poxvirus receptors are not presently known,but it is likely that there exists more than one given the ability ofpoxvirus to infect a wide range of cells. Poxvirus gene expression iswell studied due to the interest in using vaccinia virus as a vector forexpression of transgenes.

[0172] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed. Many non-viral methods ofgene transfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In particularembodiments, non-viral delivery systems rely on endocytic pathways forthe uptake of the nucleic acid molecule by the targeted cell. Exemplarynucleic acid delivery systems of this type include liposomal derivedsystems, poly-lysine conjugates, and artificial viral envelopes.

[0173] In particular embodiments, plasmid vectors are used in thepractice of the present invention. Naked plasmids can be introduced intomuscle cells by injection into the tissue. Expression can extend overmany months, although the number of positive cells is typically low(Wolff et al., (1989) Science 247:247). Cationic lipids have beendemonstrated to aid in introduction of DNA into some cells in culture(Feigner and Ringold, (1989) Nature 337:387). Injection of cationiclipid plasmid DNA complexes into the circulation of mice has been shownto result in expression of the DNA in lung (Brigham et al., (1989) Am.J. Med. Sci. 298:278). One advantage of plasmid DNA is that it may beintroduced into non-replicating cells.

[0174] In a representative embodiment, a nucleic acid molecule (e.g., aplasmid) may be entrapped in a lipid particle bearing positive chargeson its surface and, optionally, tagged with antibodies against cellsurface antigens of the target tissue (Mizuno et al., (1992) No ShinkeiGeka 20:547; PCT publication WO 91/06309; Japanese patent application1047381; and European patent publication EP-A-43075).

[0175] Liposomes that consist of amphiphilic cationic molecules areuseful non-viral vectors for nucleic acid delivery in vitro and in vivo(reviewed in Crystal, Science 270: 404-410.(1995); Blaese et al., CancerGene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5:.382-389(1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); and Gao etal., Gene Therapy 2: 710-722 (1995)). The positively charged liposomesare believed to complex with negatively charged nucleic acids viaelectrostatic interactions to form lipid:nucleic acid complexes. Thelipid:nucleic acid complexes have several advantages as gene transfervectors. Unlike viral vectors, the lipid:nucleic acid complexes can beused to transfer-expression cassettes of essentially unlimited size.Since the complexes lack proteins, they may evoke fewer immunogenic andinflammatory responses. Moreover, they cannot replicate or recombine toform an infectious agent and have low integration frequency. A number ofpublications have demonstrated that amphiphilic cationic lipids canmediate nucleic acid delivery in vivo and in vitro (Feigner et al.,Proc. Natl. Acad. Sci. USA 84: 7413-17 (1987); Loeffler et al., Methodsin Enzymology 217: 599-618 (1993); Feigner et al., J. Biol. Chem. 269:2550-2561 (1994)).

[0176] Several groups have reported the use of amphiphilic cationiclipid:nucleic acid complexes for in vivo transfection both in animalsand in humans (reviewed in Gao et al., Gene Therapy 2: 710-722 (1995);Zhu et al., Science 261: 209-211 (1993); and Thierry et al., Proc. Natl.Acad. Sci. USA 92: 9742-9746 (1995)). U.S. Pat. No. 6,410,049 describesa method of preparing cationic lipid:nucleic acid complexes that haveprolonged shelf life.

[0177] IV. Production of Recombinant Lysosomal Polypeptides.

[0178] As indicated above, recombinant lysosomal polypeptides such asGAA can be produced in, and optionally purified from, cultured cells ororganisms for a variety of purposes. Methods of delivering a recombinantnucleic acid encoding a lysosomal polypeptide for therapeutic methodsare described in more detail below. The isolated nucleic acid may becarried by a delivery vector as described in the preceding section.

[0179] Those skilled in the art will appreciate that the isolatednucleic acid encoding the lysosomal polypeptide can be operablyassociated with appropriate expression control sequences, e.g.,transcription/translation control signals, which can be included in theisolated nucleic acid or by a vector backbone. For example, specificinitiation signals are generally required for efficient translation ofinserted protein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

[0180] The isolated nucleic acid can further comprise a polyadenylationsignal (e.g., a signal for polyA polymerase to add the polyA tail to the3′ end of the transcribed mRNA). It is common, however, that thepolyadenylation signal is provided by a vector backbone into which thecoding sequence is inserted (e.g., a plasmid or a recombinant viralgenome) and, therefore, in particular embodiments a polyadenylationsignal may not be present in the isolated nucleic acid molecule.

[0181] A variety of promoter/enhancer elements may be used depending onthe level and tissue-specific expression desired. The promoter can beconstitutive or inducible (e.g., the metalothionein promoter or ahormone inducible promoter), depending on the pattern of expressiondesired. The promoter may be native or foreign and can be a natural or asynthetic sequence. By foreign, it is intended that the transcriptionalinitiation region is not found in the wild-type host into which thetranscriptional initiation region is introduced. The promoter is chosenso that it will function in the target cell(s) of interest. Promotersthat function in liver (for example, liver parenchyma, e.g., alpha-1antitrypsin promoter), skeletal muscle, cardiac muscle, smooth muscle,diaphragm muscle, endothelial cells, intestinal cells, pulmonary cells(e.g., smooth muscle or epithelium), peritoneal epithelial cells andfibroblasts are preferred. The promoter may further be “specific” forthese cells and tissues, in that it may only show significant activityin the specific cell or tissue type.

[0182] The isolated nucleic acid can be operatively associated with acytomegalovirus (CMV) major immediate-early promoter, an albuminpromoter, an Elongation Factor 1-α (EF1-α) promoter, a PγK promoter, aMFG promoter, or a Rous sarcoma virus promoter. A hybrid promotercontaining the CMV major immediate-early enhancer and chicken beta-actin(CB) promoter is also suitable. It has been speculated that drivingheterologous nucleotide transcription with the CMV promoter results indown-regulation of expression in immunocompetent animals (see, e.g., Guoet al., (1996) Gene Therapy 3:802). Accordingly, it may be advantageousto operably associate the isolated nucleic acid with a modified CMVpromoter that does not result in this down-regulation of transgeneexpression.

[0183] The isolated nucleic acids of the invention can comprise two ormore coding sequences. In embodiments wherein there is more than onecoding sequence, the coding sequences may be operatively associated withseparate promoters or, alternatively, with a single upstream promoterand one or more downstream internal ribosome entry site (IRES) sequences(e.g., the picornavirus EMC IRES sequence).

[0184] In particular embodiments of the invention, the total size of theisolated nucleic acid is less than about 5, 4.8, 4.7, 4.6, 4.5, 4.3,4.2, 4, 3.8, 3.7, 3.6, 3.5, 3.2, 3 or 2.8 kb or less in length.Relatively small expression cassettes can be particularly advantageousfor delivery by AAV vectors.

[0185] An isolated nucleic acid of the invention can be introduced intoa host cell, e.g., a cell of a primary or immortalized cell line. Therecombinant cells can be used to produce the encoded polypeptide.Generally, the isolated nucleic acid is incorporated into an expressionvector (viral or nonviral as described above).

[0186] Expression vectors can be designed for expression of polypeptidesin prokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g., in thebaculovirus expression system), yeast cells or mammalian cells. Somesuitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSecI (Baldari et al., (1987) EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,(1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Baculovirus vectors available for expression of proteins incultured insect cells (e.g., Sf 9 cells) include the pAc series (Smithet al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series(Lucklow, V. A., and Summers, M. d. (1989) Virology 170:31-39).

[0187] Examples of mammalian expression vectors include pCDM8 (Seed,(1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J.6:187-195). When used in mammalian cells, the expression vector'scontrol functions are often provided by viral regulatory elements. Forexample, commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40.

[0188] In addition to the regulatory control sequences discussed above,the recombinant expression vector can contain additional nucleotidesequences. For example, the recombinant expression vector may encode aselectable marker gene to identify host cells that have incorporated thevector.

[0189] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional technique including but not limited to calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation, microinjection andviral-mediated transfection and transduction. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory manuals.

[0190] Often only a small fraction of cells (in particular, mammaliancells) integrate the foreign DNA into their genome. In order to identifyand select these integrants, a nucleic acid sequence that encodes aselectable marker (e.g., resistance to antibiotics) can be introducedinto the host cells along with the gene encoding the protein ofinterest. Preferred selectable markers include those that conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding the protein of interest or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

[0191] A. Transgenic Animals.

[0192] The lysosomal polypeptide may be produced in a non-humantransgenic animal (i.e., containing a nucleic acid introduced by humanintervention using recombinant nucleic acid techniques). Methods forgenerating non-human transgenic animals are known in the art. DNAconstructs can be introduced into the germ line of an avian or mammal tomake a transgenic animal. For example, one or several copies of theconstruct can be incorporated into the genome of an embryo by standardtransgenic techniques.

[0193] It is often desirable to express the transgenic polypeptide inthe milk of a transgenic mammal. Mammals that produce large volumes ofmilk and have long lactating periods are preferred. Preferred mammalsare ruminants, e.g., cows, sheep, camels or goats (including goats ofSwiss origin, such as the Alpine, Saanen and Toggenburg breed goats).Other preferred mammals include oxen, rabbits and pigs.

[0194] In an exemplary embodiment, a transgenic non-human animal isproduced by introducing a transgene into the germ line of the non-humananimal. Transgenes can be introduced into embryonal target cells atvarious developmental stages. Different methods are used depending onthe stage of development of the embryonal target cell. The specificline(s) of any animal used should, if possible, be selected for generalgood health, good embryo yields, good pronuclear visibility in theembryo, and good reproductive fitness.

[0195] Introduction of the transgene into the embryo can be accomplishedby any of a variety of means known in the art such as microinjection,electroporation, lipofection or a viral vector. For example, thetransgene can be introduced into a mammal by microinjection of theconstruct into the pronuclei of the fertilized mammalian egg(s) to causeone or more copies of the construct to be retained in the cells of thedeveloping mammal(s). Following introduction of the transgene constructinto the fertilized egg, the egg can be incubated in vitro for varyingamounts of time, or reimplanted into the surrogate host, or, both. Onecommon method is to incubate the embryos in vitro for about 1-7 days,depending on the species, and then reimplant them into the surrogatehost.

[0196] The progeny of the transgenically manipulated embryos can betested for the presence of the construct, e.g., by Southern blotanalysis of a segment of tissue. An embryo having one or more copies ofthe exogenous cloned construct stably integrated into the genome can beused to establish a permanent transgenic animal line carrying thetransgenic construct.

[0197] Litters of transgenically altered mammals can be assayed afterbirth for the incorporation of the construct into the genome of theoffspring. This can be done by hybridizing a probe corresponding to thetransgenic sequence to chromosomal material from the progeny. Thosemammalian progeny found to contain at least one copy of the, constructin their genome are grown to maturity. The female species of theseprogeny will produce the desired polypeptide in or along with theirmilk. The transgenic mammals can be bred to produce other transgenicprogeny useful in producing the desired polypeptides in their milk.

[0198] Transgenic females may be tested for polypeptide secretion intomilk, using any art-known assay technique, e.g., a Western blot orenzymatic assay.

[0199] Useful transcriptional promoters for expressing the polypeptidein the milk of a transgenic animal are those promoters that arepreferentially activated in mammary epithelial cells, includingpromoters that control the genes encoding milk polypeptides such ascaseins, beta-lactoglobulin (Clark et al., (1989) Bio/Technology7:487-492), whey acid protein (Gorton et al. (1987) Bio/Technology 5:1183-1187), and lactalbumin (Soulier et al., (19192) FEBS Letts.297:13). The alpha-, beta-, gamma- or kappa-casein gene promoters of anymammalian species can be used to provide mammary expression; a preferredpromoter is the goat beta-casein gene promoter (DiTullio, (1992)Bio/Technology 10:74-77). Other milk-specific polypeptide promoter orpromoters that are specifically activated in mammary tissue can beisolated from cDNA or genomic sequences.

[0200] DNA sequence information is available for mammary gland specificgenes listed above, in at least one, and often in several organisms.See, e.g., Richards et al., J Biol Chem. 256, 526-532 (1981) (ratalpha-lactalbumin); Campbell et al., Nucleic Acids Res. 12, 8685-8697(1984) (rat WAP); Jones et al., J. Biol. Chem. 260: 7042-7050 (1985)(rat beta-casein); Yu-Lee & Rosen, J. Biol. Chem. 258, 10794-10804(1983) (rat gamma-casein); Hall, Biochem. J. 242, 735-742 (1987) (humanalpha-lactalbumin); Stewart, Nucleic Acids Res. 12, 389 (1984) (bovinealpha S1 and kappa casein cDNAs); Gorodetsky et al., Gene 66, 87-96(1-988) (bovine beta-casein); Alexander et al., Eur. J. Biochem. 178,395-401 (1988) (bovine kappa-casein); Brignon et al., FEBS Lett. 188,48-55 (1977) (bovine alpha S2 casein); Jamieson et al., Gene 61, 85-90(1987), Ivanov et al., Biol. Chem. Hoppe-Seyler 369, 425-429 (1988),Alexander et al., Nucleic Acids Res. 17, 6739 (1989) (bovine betalactoglobulin); Vilotte et al., Biochimie 69, 609-620 (1987) (bovinealpha-lactalbumin). The structure and function of the various milkprotein genes are reviewed by Mercier & Vilotte, J. Dairy Sci. 76,3079-3098 (1993). If additional flanking sequence are useful inoptimizing expression, such sequences can be cloned using the existingsequences, as probes. Mammary-gland specific regulatory sequences fromdifferent organisms can be obtained by screening libraries from suchorganisms using known cognate nucleotide sequences, or antibodies tocognate polypeptides, as probes.

[0201] According to this embodiment, the isolated nucleic acid may beoperatively associated with a milk-specific signal sequence, e.g., froma gene which encodes a product secreted into milk. For example, signalsequences from genes coding for caseins (e.g., alpha-, beta-, gamma- orkappa-caseins), beta-lactoglobulin, whey acid protein, and lactalbuminare useful in the present invention.

[0202] The polypeptide can be expressed from an illustrative expressionconstruct that includes a promoter specific for mammary epithelialcells, e.g., a casein promoter (for example, a goat beta-caseinpromoter), a milk-specific signal sequence, e.g., a casein signalsequence (for example, beta-casein signal sequence), and a sequenceencoding the polypeptide.

[0203] The transgenic polypeptide can be produced in milk at relativelyhigh concentrations and in large volumes, providing continuous highlevel output of normally processed polypeptide that is easily harvestedfrom a renewable resource. There are several different methods known inthe art for isolation of polypeptides from milk.

[0204] Milk polypeptides usually are isolated by a combination ofprocesses. Raw milk first is fractionated to remove fats, for example,by skimming, centrifugation, sedimentation (H. E. Swaisgood,Developments in Dairy Chemistry, I: Chemistry of Milk Protein, AppliedScience Publishers, NY, 1982), acid precipitation (U.S. Pat. No.4,644,056) or enzymatic coagulation with rennin or chymotrypsin(Swaisgood, ibid.). Next, the major milk proteins may be fractionatedinto either a clear solution or a bulk precipitate from which thespecific protein of interest may be readily purified.

[0205] U.S. Ser. No. 08/648,235 discloses a method for isolating asoluble milk component, such as a protein, in its biologically activeform from whole milk or a milk fraction by tangential flow filtration.Unlike previous isolation methods, this eliminates the need for a firstfractionation of whole milk to remove fat and casein micelles, therebysimplifying the process and avoiding losses of recovery and bioactivity.This method may be used in combination with additional purificationsteps to further remove contaminants and purify the component ofinterest.

[0206] B. Production of Transgenic Polypeptides in the Eggs of aTransgenic Avian.

[0207] Recombinant polypeptide can also be produced in the eggs of atransgenic avian, e.g., a transgenic chicken, turkey, duck, goose,ostrich, guinea fowl, peacock, partridge, pheasant, pigeon, quail usingmethods known in the art (Sang et al., Trends Biotechnology, 12:415-20,1994). Genes encoding polypeptides specifically expressed in the egg,such as yolk-protein genes and albumin-protein genes, can be modified todirect expression of the lysosomal polypeptides of the invention.

[0208] Useful promoters for producing polypeptides in avian eggs arethose promoters that are preferentially activated in the egg, includingpromoters that control the genes encoding egg polypeptides, e.g.,ovalbumin, lysozyme and avidin. Promoters from the chicken ovalbumin,lysozyme or avidin genes are preferred. Egg-specific promoters or thepromoters that are specifically activated in egg tissue can be from cDNAor genomic sequences.

[0209] DNA sequences of egg specific genes are known in the art (see,e.g., Burley et al., “The Avian Egg”, John Wiley and Sons, p. 472, 1989,the contents of which are incorporated herein by reference). Eggspecific regulatory sequences from different organisms can be obtainedby screening libraries from such organisms using known cognatenucleotide'sequences, or antibodies to cognate polypeptides, as probes.

[0210] C. Transgenic Plants.

[0211] Recombinant polypeptides can be expressed in a transgenic plantin which the transgene is inserted into the nuclear or plastidic genome.Plant transformation is known as the art. See, in general, Methods inEnzymology Vol. 153 (“Recombinant DNA Part D”) 1987, Wu and GrossmanEds., Academic Press and European Patent Application EP 693 554.

[0212] Foreign nucleic acids can be introduced into plant cells orprotoplasts by several methods. For example, nucleic acid can bemechanically transferred by microinjection directly into plant cells byuse of micropipettes. Foreign nucleic acid can also be transferred intoa plant cell by using polyethylene glycol which forms a precipitationcomplex with the genetic material that is taken up by the cell(Paszkowski et al. (1984) EMBO J. 3:2712-22). Foreign nucleic acid canalso be introduced into a plant cell by electroporation (Fromm et al.(1985) Proc. Natl. Acad. Sci. USA 82:5824). In this technique, plantprotoplasts are electroporated in the presence of plasmids or nucleicacids containing the relevant genetic construct. Electrical impulses ofhigh field strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form a plant callus. Selection of thetransformed plant cells with the transformed gene can be accomplishedusing phenotypic markers.

[0213] Cauliflower mosaic virus (CaMV) can be used as a vector forintroducing foreign nucleic acids into plant cells (Hohn et al. (1982)“Molecular Biology of Plant Tumors,” Academic Press, New York, pp.549-560; Howell, U.S. Pat. No. 4,407,956). CaMV viral DNA genome isinserted into a parent bacterial plasmid creating a recombinant DNAmolecule which can be propagated in bacteria. The recombinant plasmidcan be further modified by introduction of the desired DNA sequence. Themodified viral portion of the recombinant plasmid is then excised fromthe parent bacterial plasmid, and used to inoculate the plant cells orplants.

[0214] High velocity ballistic penetration by small particles can beused to introduce foreign nucleic acid into plant cells. Nucleic acid isdisposed within the matrix of small beads or particles, or on thesurface (Klein et al. (1987) Nature 327:70-73). Although typically onlya single introduction of a new nucleic acid segment is required, thismethod also provides for multiple introductions.

[0215] A nucleic acid can be introduced into a plant cell by infectionof a plant cell, an explant, a meristem or a seed with Agrobacteriumtumefaciens or Agrobacterium rhizogenes transformed with the nucleicacid. Under appropriate conditions, the transformed plant cells aregrown to form shoots, roots, and develop further into plants. Thenucleic acids can be introduced into plant cells, for example, by meansof the Ti plasmid of Agrobacteria. The Ti plasmid is transmitted toplant cells upon infection by Agrobacteria, and is stably integratedinto the plant genome (Horsch et al. (1984) “Inheritance of FunctionalForeign Genes in Plants,” Science 233:496498; Fraley et al. (1983) Proc.Natl. Acad. Sci. USA 80:4803).

[0216] Plants from which protoplasts can be isolated and cultured togive whole regenerated plants can be transformed so that whole plantsare recovered which contain the transferred foreign gene. Some suitableplants include, for example, species from the genera Fragaria, Lotus,Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum,Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis,Atropa, Capsicum, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciohorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium,Zea, Triticum, Sorghum, and Datura.

[0217] Plant regeneration from cultured protoplasts is described inEvans et al., “Protoplasts Isolation and Culture,” Handbook of PlantCell Cultures 1:124-176 (MacMillan Publishing Co. New York 1983); M. R.Davey, “Recent Developments in the Culture and Regeneration of PlantProtoplasts,” Protoplasts (1983)-Lecture Proceedings, pp.12-29,(Birkhauser, Basal 1983); P. J. Dale, “Protoplast Culture and PlantRegeneration of Cereals and Other Recalcitrant Crops,” Protoplasts(1983)-Lecture Proceedings, pp. 31-41, (Birkhauser, Basel 1983); and H.Binding, “Regeneration of Plants,” Plant Protoplasts, pp. 21-73, (CRCPress, Boca Raton 1985).

[0218] Regeneration from protoplasts varies from species to species ofplants, but generally a suspension of transformed protoplasts containingcopies of the exogenous sequence is first generated. In certain species,embryo formation can then be induced from the protoplast suspension, tothe stage of ripening and germination as natural embryos. The culturemedia can contain various amino acids and hormones, such as auxin andcytokinins. It can also be advantageous to add glutamic acid and prolineto the medium, especially for such species as corn and alfalfa. Shootsand roots normally develop simultaneously. Efficient regeneration willdepend on the medium, on the genotype, and on the history of theculture. If these three variables are controlled, then regeneration isfully reproducible and repeatable.

[0219] In vegetatively propagated crops, the mature transgenic plantscan be propagated by the taking of cuttings or by tissue culturetechniques to produce multiple identical plants for trialling, such astesting for production characteristics. Selection of a desirabletransgenic plant is made and new varieties are obtained thereby, andpropagated vegetatively for commercial sale. In seed propagated crops,the mature transgenic plants can be self crossed to produce a homozygousinbred plant. The inbred plant produces seed containing the transgene.These seed can be grown to produce plants that have the selectedphenotype. The inbreds according to this invention can be used todevelop new hybrids. In this method a selected inbred line is crossedwith another inbred line to produce the hybrid.

[0220] Parts obtained from a transgenic plant, such as flowers, seeds,leaves, branches, fruit, and the like are covered by the invention,provided that these parts include cells which have been so transformed.Progeny and variants, and mutants of the regenerated plants are alsoincluded within the scope of this invention, provided that these partscomprise the introduced nucleic acid sequences. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof this invention.

[0221] Selection of transgenic plants or plant cells can be based upon avisual assay, such as observing color changes (e.g., a white flower,variable pigment production, and uniform color pattern on flowers orirregular patterns), but can also involve biochemical assays of eitherenzyme activity or product quantitation. Transgenic plants or plantcells are grown into plants bearing the plant part of interest and thegene activities are monitored, such as by biochemical assays (Northernblots); Western blots; and enzyme assays, Appropriate plants areselected and further evaluated. Methods for generation of geneticallyengineered plants are further described in U.S. Pat. Nos. 5,283,184,5,482,852, and European Patent Application EP 693 554, all of which areincorporated herein by reference.

[0222] V. Subjects, Pharmaceutical Formulations, Vaccine and Modes ofAdministration.

[0223] The present invention finds use in veterinary and medicalapplications. Suitable subjects include both avians and mammals, withmammals being preferred. The term “avian” as used herein includes, butis not limited to, chickens, ducks, geese, quail, turkeys and pheasants.The term “mammal” as used herein includes, but is not limited to,humans, non-human primates, bovines, ovines, caprines, equines, felines,canines, lagomorphs, rats, mice etc. Human subjects are preferred. Humansubjects include neonates, infants, juveniles, and adults. Inrepresentative embodiments, the subject is a human subject that has oris believed to have a lysosomal polypeptide (e.g., GAA) deficiency.

[0224] In particular embodiments, the present invention provides apharmaceutical composition comprising an isolated nucleic acid or vectorof the invention in a pharmaceutically-acceptable carrier and,optionally, other medicinal agents, pharmaceutical agents, carriers,adjuvants, dispersing agents, diluents, and the like. For injection, thecarrier will typically be a liquid, such as sterile pyrogen-free water,pyrogen-free phosphate-buffered saline solution, bacteriostatic water,or Cremophor EL[R] (BASF, Parsippany, N.J.). For other methods ofadministration, the carrier may be either solid or liquid.

[0225] By “pharmaceutically acceptable” it is meant a material that isnot biologically or otherwise undesirable, i.e., the material may beadministered to a subject along with the isolated nucleic acid or vectorwithout causing any undesirable biological effects such as toxicity.Thus, such a pharmaceutical composition can be used, for example, intransfection of a cell ex vivo or in administering an isolated nucleicacid or vector directly to a subject.

[0226] In the case of a viral vector, virus particles may be contactedwith the cells at the appropriate multiplicity of infection according tostandard transduction methods appropriate for the particular targetcells. Titers of virus to administer can vary, depending upon the targetcell type and the particular virus vector, and can be determined bythose of skill in the art without undue experimentation. Typically, atleast about 10³ virus particles, at least about 10⁵ particles, at leastabout 10⁷ particles, at least about 10⁹ particles, at least about 10¹¹particles, or at least about 10¹² particles are administered to thecell. In exemplary embodiments, about 10⁷ to about 10¹⁵ particles, about10⁷ to about 10¹³ particles, about 10⁸to about 10¹² particles, about10¹⁰ to about 10¹⁵ particles, about 10¹¹ to about 10¹⁵ particles, about10¹² to about 10¹⁴ particles, or about 10¹² to about 10¹³ particles areadministered.

[0227] The cell to be administered the vectors of the invention can beof any type, including but not limited to neuronal cells (includingcells of the peripheral and central nervous systems), retinal cells,epithelial cells (including dermal, gut, respiratory, bladder,pulmonary, peritoneal and breast tissue epithelium), muscle (includingcardiac, smooth muscle, including pulmonary smooth muscle cells,skeletal muscle, and diaphragm muscle), pancreatic cells (includingislet cells), hepatic cells (including parenchyma), cells of theintestine, fibroblasts (e.g., skin fibroblasts such as human skinfibroblasts), fibroblast-derived cells, endothelial cells, intestinalcells, germ cells, lung cells (including bronchial cells and alveolarcells), prostate cells, stem cells, progenitor cells, dendritic cells,and the like. Alternatively, the cell is a cancer cell (including tumorcells). Moreover, the cells can be from any species of origin, asindicated above.

[0228] Mammalian cells include but are not limited to CHO cells, 293cells, HT1080 cells, HeLa cells or C10 cells.

[0229] In particular embodiments of the invention, the cell has beenremoved from a subject, the vector is introduced therein, and the cellis then replaced back into the subject. Methods of removing cells fromsubjects for treatment ex vivo, followed by introduction back into thesubject are known in the art (see,. e.g., U.S. Pat. No. 5,399,346 forthe teaching of ex vivo virus vector administration). As a furtheralternative, the cells that are manipulated and then introduced into thesubject are provided from another subject or cell line as a cell-basedform of therapy.

[0230] A further aspect of the invention is a method of treatingsubjects in vivo with the inventive nucleic acids or delivery vectors.Administration of the nucleic acid or delivery vectors of the presentinvention to a human subject or an animal can be by any means known inthe art. The subject can be a mammalian subject, more particularly ahuman subject. In other embodiments, the subject is in need oftreatment, for example, has been diagnosed with or is suspected ofhaving a lysosomal polypeptide (e.g., GAA) deficiency.

[0231] Dosages will depend upon the mode of administration, the severityof the disease or condition to be treated, the individual subject'scondition, the particular vector, and the gene to be delivered, and canbe determined in a routine manner (see, e.g., Remington, The Science AndPractice of Pharmacy (9^(th) Ed. 1995)). In particular embodiments, theisolated nucleic acid or vector is administered to the subject in atherapeutically effective amount, as that term is defined above.

[0232] Typically, with respect to viral vectors, at least about 10³, atleast about 10⁵, at least about 10⁷, at least about 10⁹, at least about10¹¹ virus particles, or at least about 10¹² virus particles areadministered to the subject per treatment. Exemplary doses are virustiters of about 10⁷ to about 10¹⁵ particles, about 10⁷ to about 10¹⁴particles, about 10⁸to about 10¹³ particles, about 10¹⁰ to about 10¹⁵particles, about 10¹¹ to about 10¹⁵ particles, about 10¹² to about10¹⁴particles, or about 10¹²to about 10¹³ particles.

[0233] In particular embodiments of the invention, more than oneadministration (e.g., two, three, four, or more administrations) may beemployed to achieve therapeutic levels of nucleic acid expression.

[0234] Exemplary modes of administration include oral, rectal,transmucosal, topical, transdermal, inhalation, parenteral, e.g.,intravenous, subcutaneous, intradermal, intramuscular (i.e.,administration to cardiac, skeletal, diaphragm and/or smooth muscle),and intraarticular administration, and the like, as well as directtissue (e.g., muscle) or organ injection (e.g., into the liver, into thebrain for delivery to the central nervous system), alternatively,intrathecal, direct intramuscular (e.g., into cardiac, skeletal, ordiaphragm muscle), intraventricular, intravenous, intraperitoneal,intranasal, or intraocular injections. Administration to the liver(discussed below) is another representative mode of administration.

[0235] Injectables can be prepared in conventional forms, either as,liquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. An injectionmedium will typically be an aqueous liquid that contains the additivesusual for injection solutions, such as stabilizing agents, salts orsaline, and/or buffers.

[0236] For oral administration, the isolated nucleic acid or vector canbe administered in solid dosage forms, such as capsules, tablets, andpowders, or in liquid dosage forms, such as elixirs, syrups, andsuspensions. Active component(s) can be encapsulated in gelatin capsulestogether with inactive ingredients and powdered carriers, such asglucose, lactose, sucrose, mannitol, starch, cellulose or cellulosederivatives, magnesium stearate, stearic acid, sodium saccharin, talcum,magnesium carbonate and the like. Examples of additional inactiveingredients that may be added to provide desirable color, taste,stability, buffering capacity, dispersion or other known desirablefeatures are red iron oxide, silica gel, sodium lauryl sulfate, titaniumdioxide, edible white ink and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

[0237] Oral administration of AAV vectors has been described by U.S.Pat. No. 6,110,456 (incorporated by reference herein in its entirety).

[0238] The isolated nucleic acid or vector may alternatively beformulated for nasal administration or otherwise administered to thelungs of a subject by any suitable means, but is preferably administeredby an aerosol suspension of respirable particles comprising the vector,which the subject inhales. The respirable particles may be liquid orsolid. The term “aerosol” includes any gas-borne suspended phase, whichis capable of being inhaled into the bronchioles or nasal passages.Specifically, aerosol includes a gas-borne suspension of droplets, asmay be produced in a metered dose inhaler or nebulizer, or in a mistsprayer. Aerosol also includes a dry powder composition suspended in airor other carrier gas, which may be delivered by insufflation from aninhaler device, for example. See Ganderton & Jones, Drug Delivery to theRespiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviewsin Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al. (1992)J. Pharmacol. Toxicol. Methods 27:143-159. Aerosols of liquid particlescomprising the isolated nucleic acid or vector may be produced by anysuitable means, such as with a pressure-driven aerosol nebulizer or anultrasonic nebulizer, as is known to those of skill in the art. See,e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprisingthe isolated nucleic acid or vector may likewise be produced with anysolid particulate medicament aerosol generator, by techniques known inthe pharmaceutical art.

[0239] Alternatively, one may administer the isolated nucleic acid orvector in a local rather than systemic manner, for example, in a depotor sustained-release formulation.

[0240] In particular embodiments of the invention, the isolated nucleicacid or vector is delivered to the liver of the subject. Administrationto the liver can be achieved by any method known in the art, including,but not limited to intravenous administration, intraportaladministration, intrabiliary administration, intra-arterialadministration, injection into the liver parenchyma, and intrasplenicinjection.

[0241] Intramuscular delivery and intracardiac delivery to skeletalmuscle or cardiac muscle, respectively, or direct injection intodiaphragm muscle is also preferred. In other particular embodiments,intraperitoneal administration is used to deliver the isolated nucleicacid or vector to diaphragm muscle.

[0242] In particular embodiments, the isolated nucleic acid (e.g.,carried by an Ad, AAV or hybrid Ad/AAV vector) encoding a lysosomalpolypeptide is introduced into a depot organ or tissue (e.g., liver,skeletal muscle, lung) and the polypeptide is expressed therein andsecreted into the circulatory system, where it is optionally deliveredto target tissues, preferably, in a therapeutically effective amount.Intramuscular delivery to skeletal muscle or delivery to the liver areillustrative for the practice of this embodiment of the invention.Alternatively, the isolated nucleic acid or vector can be administeredto the brain (e.g., to treat MPS disorders such as Sly disease), wherethe polypeptide can be expressed and secreted by transformed ortransduced cells (e.g., neurons, glial cells) and taken up by otherbrain cells.

[0243] Having now described the invention, the same will be illustratedwith reference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

EXAMPLE 1 Materials and Methods

[0244] Cell Culture. 293 cells and C-7 cells (Amalfitano andChamberlain. (1997) Gene Ther. 4:258-263) were maintained in Dulbecco'smodified Eagle medium supplemented with 10% fetal bovine serum, 100 Upenicillin per milliliter, and 100 μg streptomycin per milliliter at 37°C. in a 5% CO₂-air atmosphere. C-7 cells were grown in the presence ofhygromycin, 50 μg/ml. HeLa cells were maintained in minimum essentialmedium Eagle supplemented with 10% fetal bovine, serum, 1 mM minimumessential medium sodium pyruvate, 0.1 mM minimum-essential mediumnonessential amino acids, 100 U penicillin per milliliter, and 100 μgstreptomycin per milliliter at 37° C. in a 5% CO₂-air atmosphere,

[0245] Construction of an AAV Vector Plasmid Encoding hGAA. The hGAAcDNA was subcloned with the CMV promoter from pcDNA3-hGAA (Van Hove etal. (1996). Proc. Natl. Acad. Sci. USA 93:65-70) into an AAV vectorplasmid, as an Nrul-EcoRV fragment, upstream of the human growth hormoneintron 4 and polyadenylation sequence (Brinster et al. (1988). Proc.Natl. Acad. Sci. USA 85: 836-840). The resulting transcriptional unitwas flanked by the AAV2 terminal repeat (TR) sequences in pAAV-ChGAAGH.A 530 bp deletion spanning the human growth hormone intron 4 wasgenerated by EcoRV and partial PvuII digestion followed by blunting ofends with T4 DNA polymerase and ligation with T4 DNA ligase to generatepAAV-ChGAAG(−). The hybrid CMV enhancer/chicken β-actin (CB) promoterwas amplified by polymerase chain reaction from pTriEx1 (Novagen,Madison, Wis.) with primers that introduced unique upstream XbaI anddownstream KpnI restriction sites, and the CB promoter was subcloned asa KpnI-XbaI fragment to replace the CMV promoter in pAAV-ChGAAG(−), togenerate pAAV-CBhGAAG(−). Next, in order to reduce the packaging sizefurther, the plasmid pAAV-CBhGAAG(−) was linearized at a unique AflIIsite in the 3′ untranslated sequence of the hGAA cDNA and partiallydigested with NspI to introduce a 411 bp deletion in the 3′ untranslatedsequence of the hGAA cDNA, followed by blunting of ends with T4 DNApolymerase and ligation with T4 DNA ligase to generate pAAV-CBGAApA.Finally, the vector sequences from pAAV-CBGAApA were isolated as a 4.4kbp fragment from a partial BglII digest, and ligated with the calfintestinal alkaline phosphatase-dephosphorylated BglII site of pShuttle(He et al. (1998). Proc. Natl. Acad. Sci. USA 95:2509-2514).

[0246] Construction of a hybrid [E1-,polymerase-, preterminal protein-]Ad-AAV vector encoding hGAA. Kanamycin-resistant shuttle plasmids wereconstructed to contain within the Ad E1 region the CB promoter+hGAAcDNA+polyA transgene cassette flanked by the AAV2 TR sequences. Theshuttle plasmid was digested with PmeI, and electroporated into theBJ5183 recombinogenic strain of E. Coli with the pAd[E1-, polymerase-,preterminal protein-] plasmid (Hodges et al. (2000). J. Gene Med.2:250-259). Recombinant kanamycin-resistant clones were screened byrestriction enzyme digestion (BstXI) to confirm successful generation ofthe full-length recombinant Ad vector genomes. These clones weredigested with PacI and transfected as previously described into the E1,and E2b expressing cell line, C-7 (Hodges et al. (2000). J. Gene Med.2:250-259). The vectors were amplified and confirmed to have the correctconstruction by restriction enzyme mapping of vector genomes, andsubsequent functional assays in vitro and in vivo. Once isolated, therespective Ad vectors are serially propagated in increasing numbers ofC-7 cells (Amalfitano et al. (1998) J. Virol. 72:926-933). Forty-eighthours after infection, infected cell pellets were harvested by low speedcentrifugation, resuspended in 10 mM Tris-HCl pH 8.0, vector releasedfrom the cells by repeated freeze-thawing (×3) of the lysate, releasedby ultrasonification, and the vector containing supernatant subjected totwo rounds of equilibrium density CsCl centrifugation (Amalfitano et al.(1998) J. Virol. 72:926-933). Two virus bands were visible. The virusbands were then removed, dialyzed extensively against 10 mM Tris-HCl pH8.0 (or PBS), sucrose added to 1%, and aliquots stored at −80° C. Thenumber of vector particles was quantified based on the OD₂₆₀ of vectorcontained in dialysis buffer with sodium dodecyl sulfate (SDS)disruption, and by DNase I digestion, DNA extraction, and Southern blotanalysis.

[0247] Hybrid Ad-AAV vector DNA analysis consisted of vector DNAisolation and restriction enzyme digestion followed by Southern blottingto verify the presence of intact AAV vector sequences within the lowerband in the cesium chloride gradient, including restriction enzymes thatdemonstrated the presence of AAV terminal repeat sequences flanking thetransgene (AhdI and BssHII).

[0248] All viral vector stocks were handled according to BiohazardSafety Level 2 guidelines published by the NIH.

[0249] In vivo administration of hybrid Ad-AAV or AAV vector stocks. Thevector was administered intramuscularly into both gastrocnemius musclesof 3-day-old GAA-KO mice (Raben et al. (1998) J. Biol. Chem.273:19086-19092). A total of 4×10¹⁰ DNase l-resistant Ad-AAV vectorparticles were administered, divided between 2 equal injections peranimal. Alternatively, a total of 10¹¹ DNase I-resistant AAV vectorparticles was administered intramuscularly in the right gastrocnemiusmuscle of 6 week-old GAA-KO/SCID mice. For liver-targetedadministration, a total of 10¹¹ DNase I-resistant AAV vector particleswas administered intravenously via the retroorbital sinus or via theportal vein to 3 month-old GAA-KO/SCID or GAA-KO mice as indicated. Atthe respective time points post-injection, plasma or tissue samples wereobtained and processed as described below. All animal procedures weredone in accordance with Duke University Institutional Animal Care andUse Committee-approved guidelines.

[0250] Determination of hGAA activity and glycogen content Tissue hGAAactivity was measured following removal of the tissue from control ortreated mice, flash-freezing on dry ice, homogenization and sonicationin distilled water, and pelleting of insoluble membranes/proteins bycentrifugation. The protein concentrations of the clarified suspensionswere quantified via the Bradford assay. hGAA activity in the muscle wasdetermined as described (Amalfitano et al. (1999) Proc. Natl. Acad. Sci.USA 96:8861-8866). Glycogen content of tissues was measured using theAspergillus niger assay system, as described (Kikuchi et al. (1998) J.Clin. Invest. 101:827-833). A two-tailed homscedastic Student's t-testwas used to determine significant differences in hGAA levels andglycogen content between GAA-KO mice with or without administration ofthe vector encoding hGAA.

[0251] Western blotting analysis of hGAA. For direct detection of hGAAin tissues, samples (100 μg of protein) were electrophoresed overnightin a 6% polyacrylamide gel to separate proteins, and transferred to anylon membrane. The blots were blocked with 5% nonfat milk solution,incubated with primary and secondary antibodies and visualized via theenhanced chemiluminescence (ECL) detection system (Amersham Pharmacia,Piscataway, N.J.).

[0252] ELISA detection of plasma anti-hGAA and anti-Ad antibodies.Recombinant hGAA (5 μg) in carbonate buffer was coated onto each well ofa 96-well plate at 4° C. overnight. Alternatively, the wells were coatedwith 5×10⁸ Ad vector particles per well at 4° C. overnight for detectionof anti-Ad antibody. After washing with phosphate buffered saline (PBS)containing 0.05% Tween 20, serial dilutions of the plasma were added tothe wells, and incubated for 1 hour at room temperature. The wells werewashed with 0.05% Tween 20+-PBS, incubated with a 1:2,500 dilution ofalkaline phosphatase-conjugated sheep anti-mouse IgG (H+L) at roomtemperature for 1 hour, washed, and alkaline phosphatase substrate(p-nitrophenyl phosphate) added. The absorbance values of the plateswere read at 405 nm with a Bio-Rad microplate reader ELISA (all samplesyielded absorbance values that were within the linear range of the assayat this dilution) (Ding et al. (2002) Mol. Ther. 5:436-446). The titerof antibody was determined as the highest dilution where the value forabsorbance exceeded 0.1.

Example 2 Neonatal Muscle-Targeted Ad-AAV Administration and hGAAProduction in GAA-KO Mice

[0253] Muscle-targeted expression of hGAA with an Ad-AAV hybrid vectorwas evaluated in neonatal GAA-KO mice. While the Ad-AAV vector deliveredmuscle-targeted hGAA in these experiments, the hybrid Ad-AAV vector inquestion was developed to provide improved AAV vector packagingefficiency. Administration of the Ad-AAV vector in 3 day-old micereversed the effects of GSD II within the injected gastrocnemius muscle,and sustained hGAA expression provided long-term therapeutic resultsevidenced by generally reduced glycogen storage in the muscles of thehind limb.

[0254] The Ad-AAV vector encoding hGAA was targeted to bothgastrocnemius muscles by intramuscular injection on day of life 3 inGAA-KO mice, and hGAA levels were analyzed at 6,12, and 24 weeks of age.Western blot analysis of hGAA in the gastrocnemius, hamstrings andquadriceps muscle groups at, 24 weeks of age showed hGAA of the ˜110 kD,76 kD and 67 kD isoforms following Ad-AAV vector administration, andhGAA was absent for the skeletal muscle of untreated GAA-KO mice (FIG. 1Pan I A). Apparently due to transduction of muscle adjacent to thegastrocnemius, the highest amount of hGAA was seen in the hamstrings,with slightly lower hGAA in the gastrocnemius and the lowest hGAA in thequadriceps. A similar pattern of introduced hGAA was detected by Westernblot analysis of gastrocnemius and quadriceps muscle groups followingAd-AAV vector administration at 6 and 12 weeks of age (not shown).

[0255] Western blot analysis of hGAA in heart, diaphragm, and liver at6, 12, and 24 weeks of age following neonatal Ad-AAV vectoradministration demonstrated low, detectable levels of hGAA for the ˜76kD isoform of hGAA in 2 of 4 GAA-KO mice at 6 weeks (FIG. 1 Panel B; m1and m4). Lower hGAA was present in the heart for 1 of 4 GAA-KO mice at24 weeks (FIG. 1 Panel B; m12), and was not detected in the heart for 5GAA-KO mice at 12 weeks of age (FIG. 1 Panel B) Finally, hGAA was notdetected in plasma by Western blot at 3 weeks following vectoradministration (not shown).

[0256] The function of hGAA introduced by the Ad-AAV vector was analyzedby GAA enzyme assay, and GAA activity correlated with the relativeamounts of hGAA protein detected by Western blot analysis (FIG. 2 PanelA). At 24 weeks, the highest GAA activity was present in hamstrings(1180±620 nmol/mg/hr), followed by gastrocnemius (717±275 nmol/mg/hr),and lowest in quadriceps (44±28 nmol/mg/hr). GAA activity was elevatedin all muscle groups at 6, 12, and 24 weeks, compared to the same musclegroups in untreated, GAA-KO mice (FIG. 2 Panel B). The hGAA activity forhamstrings was approximately 50-fold elevated compared to the GAAactivity in wild-type mouse skeletal muscle (Ding, E., et al. (2002)Mol. Ther. 5:436-446).

[0257] GAA activity was elevated in heart, diaphragm and liver at 6weeks of age following Ad-AAV vector administration in 2 of 4 GAA-KOmice (FIG. 2 Panel B; ml and m4). However, GAA activity in these tissueswas much lower than for skeletal muscles in the hindleg closer to thesite of Ad-AAV vector injection (FIG. 2 Panel B).

EXAMPLE 3 Anti-hGAA Antibodies Following Neonatal Ad-AAV Administration

[0258] Anti-hGAA antibody formation occurred during hGAA enzymereplacement in GSD II during a clinical trial (Amalfitano et al. (2001)Genet. In Med. 3:132-138) and in preclinical use of adenoviral vectorsin GAA-KO mice (Ding et al. (2002) Mol. Ther. 5:436446). Anti-hGAAantibodies were detected in GAA-KO mice at 6, 12, and 24 weeks afterneonatal Ad-AAV vector administration (FIG. 3 Panel A, Neonatalintramuscular Ad-AAV), with 3 exceptions, while anti-hGAA antibodieswere absent in untreated GAA-KO mice (FIG. 3 Panel A, Control). AdultGAA-KO mice that received the Ad-AAV vector intravenously were used aspositive controls (FIG. 3 Panel A, Ad-AAV). These mice developedhigh-titer antihGAA antibodies as reported previously (Ding, E., et al.(2002) Mol. Ther. 5:436-446). The presence of hGAA antibodies at a titerof >1:4,000 was demonstrated for 10 of 13 mice following neonatal Ad-AAVvector administration (FIG. 3 Panel B, Neonatal Intramuscular Ad-AAV),and for 3 of 3 adult GAA-KO mice following intravenous Ad-AAV vectoradministration (FIG. 3 Panel B, Ad-AAV). Only the 3 GAA-KO mice thatfailed to generate anti-hGAA antibodies following neonatal vectoradministration (m1, m4, and m12 in FIG. 3 Panel B) also featuredsignificant hGAA by Western blot analysis in heart at 6 and 24 weeks ofage (FIG. 1 Panel B). The formation of anti-hGAA antibodies appeared tobe related to the persistent expression of the foreign transgene (hGAA),because GAA-KO mice failed to generate anti-Ad antibodies followingneonatal, one-time exposure to the Ad-AAV vector (FIG. 3 Panel C).

EXAMPLE 4 Reduced Glycogen Content in Skeletal Muscles of the Hind LimbFollowing Injection of the Gastrocnemius in Neonatal GAA-KO Mice

[0259] The benefit of introduced hGAA in GSD II was shown by glycogenquantitation in skeletal muscle. Glycogen storage was reducedsignificantly for skeletal muscle groups at all time points except forthe quadriceps at 24 weeks (FIG. 4 Panel A), when hGAA in the quadricepswas not as high as for other timepoints (FIG. 2 Panel A). Glycogencontent was significantly reduced for the hamstrings, gastrocnemius, andquadriceps compared to untreated, GAA-KO mice (FIG. 4 Panel A). Glycogencontent was reduced to 0.77±0.42 mmol glucose/gm protein in the heart ofGAA-KO mice (n=4) 6 weeks after neonatal Ad-AAV vector administration,compared to 2.1±1.3 mmol glucose/gm protein in untreated GAA-KO mice(n=3). Only 2 of 4 GAA-KO mice had detectable heart and diaphragm hGAAat 6 weeks by Western blot analysis (FIG. 1 Panel B; m1 and m4), andboth the heart and diaphragm glycogen content was somewhat reduced in 1of those mice (FIG. 4 Panel B; m4).

[0260] The correction of glycogen storage, by introduced hGAA wasevident by glycogen staining of gastrocnemius and heart. Periodic-acidSchiff (PAS) staining of gastrocnemius showed much less lysosomalaccumulation of glycogen at 24 weeks following Ad-AAV vectoradministration compared to an untreated GAA-KO mouse (FIG. 5). In aGAA-KO mouse that had significant hGAA in heart at 6 weeks (FIG. 1 PanelB; m4), the glycogen accumulation in heart was less than for anuntreated, GAA-KO mouse (FIG. 5). In aggregate, these data provideevidence for the continued benefit of hGAA introduced with a vector inmuscle.

EXAMPLE 5 Construction of an AAV Vector Plasmid Encoding hGAA

[0261] The hGAA cDNA was subcloned with the CMV promoter frompcDNA3-hGAA (Van Hove et al. (1996). Proc. Natl. Acad. Sci. USA93:65-70) into an AAV vector plasmid, as an Nrul-EcoRV fragment,upstream of the human growth hormone intron 4 and polyadenylationsequence (Brinster et al. (1988). Proc. Natl. Acad. Sci. USA 85:836-840). The resulting transcriptional unit was flanked by the AAV2 TRsequences in pAAV-ChGAAGH. A 530 bp deletion spanning the human growthhormone intron 4 was generated by EcoRV and partial PvuII digestionfollowed by blunting of ends with T4 DNA polymerase and ligation with T4DNA ligase to generate pAAV-ChGAAG(−). The hybrid CMV enhancer/chickenβ-actin (CB) promoter was amplified by polymerase chain reaction frompTriEx1 (Novagen, Madison, Wis.) with primers that introduced uniqueupstream XbaI and downstream KpnI restriction sites, and the CB promoterwas subcloned as a KpnI-XbaI fragment to replace the CMV promoter inpAAV-ChGAAG(−), to generate pAAV-CBhGAAG(−). In order to reduce thepackaging size further, the plasmid pAAV-CBhGAAG(−) was linearized at aunique AflII site in the 3′ untranslated sequence of the hGAA cDNA andpartially digested with NspI to introduce a 411 bp deletion in the 3′untranslated sequence of the hGAA cDNA, followed by blunting of endswith T4 DNA polymerase and ligation with T4 DNA ligase to generatepAAV-CBGAApA. Finally, the vector sequences from pAAV-CBGAApA wereisolated as a 4.4 kbp fragment from a partial BglII digest, and ligatedwith the calf intestinal alkaline phosphatase-dephosphorylated BglIIsite of pShuttle (He, T.-C., et al. (1998). Proc. Natl. Acad. Sci. USA95:2509-2514).

EXAMPLE 6 Construction of a Hybrid [E1-,Polymerase-, PreterminalProtein-] Ad-AAV Vector Encoding hGAA

[0262] Kanamycin-resistant'shuttle plasmids were constructed to containwithin the Ad E1 region the CB promoter+hGAA cDNA+polyA transgenecassette flanked by the AAV2 TR sequences. The shuttle plasmid wasdigested with PmeI, and electroporated into the BJ5183 recombinogenicstrain of E. Coli with the pAd[E1-, polymerase-, preterminal protein-]plasmid (Hodges et al. (2000). J. Gene Med. 2:250-259). Recombinantkanamycin-resistant clones were screened by restriction enzyme digestion(BstXI) to confirm successful generation of the full-length recombinantAd vector genomes. These clones were digested with PacI and transfectedas previously described into the E1, and E2b expressing cell line, C-7(Amalfitano et al. (1998). J. Virol. 72:926-933).

[0263] The vectors were amplified and confirmed to have the correctconstruction by restriction enzyme mapping of vector genomes, andsubsequent functional assays in vitro and in vivo. Once isolated, therespective Ad vectors are serially propagated in increasing numbers ofC-7 cells (Amalfitano et al. (1998). J. Virol. 72:926-933). Forty-eighthours after infection, infected cell pellets were harvested by low speedcentrifugation, resuspended in 10 mM Tris-HCl pH 8.0, vector releasedfrom the cells by repeated freeze-thawing (×3) of the lysate, releasedby ultrasonification, and the supernatant containing vector wassubjected to two rounds of equilibrium density CsCl centrifugation(Amalfitano et al. (1998). J. Virol. 72:926-933). Two virus bands werevisible. The virus bands were then removed, dialyzed extensively against10 mm Tris-HCl pH 8.0 (or PBS), sucrose added to 1%, and aliquots storedat −80° C. The number of vector particles was quantified based on theOD₂₆₀ of vector contained in dialysis buffer with sodium dodecyl sulfate[SDS] disruption, and by DNase I digestion, DNA extraction, and Southernblot analysis.

[0264] Hybrid Ad-AAV vector DNA analysis consisted of vector DNAisolation and restriction enzyme digestion followed by Southern blottingto verify the presence of intact AAV vector sequences within the lowerband in the cesium chloride gradient, including restriction enzymes thatdemonstrated the presence of AAV terminal repeat sequences flanking thetransgene (AhdI and BssHII) (Sun et al., (2003) Mol Ther 7:193-201).

EXAMPLE 7 Preparation of AAV Vectors

[0265] AAV vector stocks were prepared as described herein withmodifications as described (Sun B D, Chen Y-T, Bird A, Xu F, Hou Y-X,Amalfitano A, and Koeberl D D. Packaging of an AAV vector encoding humanacid α-glucosidase for gene therapy in glycogen storage disease type IIwith a modified hybrid adenovirus-adeno-associated virus vector. MolTher 7:467477,.2003; Halbert C L, Allen J M, Miller A D.Adeno-associated virus type 6 (AAV) vectors mediate efficienttransduction of airway epithelial cells in mouse lungs compared to thatof AAV2 vectors. (Halbert et al., (2001) J Virol 75:6615-6624). Briefly,293 cells were infected with the hybrid Ad-AAV vector (2000 DNaseI-resistant vector particles/cell as quantitated by Southern blotanalysis) containing the AAV vector sequences 15-30 minutes beforetransfection with an AAV packaging plasmid containing the AAV2 Rep andAAV2 or AAV6 (for AAV6 vector [Halbert et al. (2001) J. Virol.75:6615-6624]). Cap genes were driven by heterologous promoters, whichtypically generate no detectable replication-competent AAV (rcAAV)(Allen et al. (2000) Mol. Ther. 1:88-95; Allen et al. (1997) J. Virol.71:6816-6822). Cell lysate was harvested 48 hours following infectionand freeze-thawed 3 times, isolated by iodixanol step gradientcentrifugation before heparin affinity column purification (Zolotukhinet al. (1999) Gene Ther. 6:973-995; Halbert C L, Allen J M, Miller A D.Adeno-associated virus type 6 (AAV) vectors mediate efficienttransduction of airway epithelial cells in mouse lungs compared to thatof AAV2 vectors. J Virol 2001; 75:6615-6624.), and aliquots were storedat −80° C. The number of vector DNA containing-particles was determinedby DNase I digestion, DNA extraction, and Southern blot analysis.Contaminating wt AAV particles were detected in recombinant AAV vectorpreparations by Southern blot analysis of extracted vector DNA, and by asensitive PCR assay utilizing primers spanning the junction between therep and cap genes. The level of rcAAV was less than 1 particle in 10⁵AAV vector particles. All viral vector stocks were handled according toBiohazard Safety Level 2 guidelines published by the NIH.

EXAMPLE 8 In vivo -Administration of AAV Vector Stocks

[0266] AAV vector was administered intramuscularly into thegastrocnemius muscle of 6 week-old GAA-KO mice (Raben et al. (1998) J.Biol. Chem. 273:19086-19092)/SCID mice. One hundred μl containing 1×10¹¹DNase I-resistant AAV vector particles were injected per gastrocnemius.For portal vein injection, an AAV vector was administered via portalvein injection. At the respective time points post-injection, plasma ortissue samples were obtained and processed as described below. Forintravenous administration, an AAV vector was administered via theretroorbital sinus. All animal procedures were done in accordance withDuke University Institutional Animal Care and Use Committee-approvedguidelines.

EXAMPLE 9 Determination of hGAA Activity and Glycogen Content

[0267] Tissue hGAA activity was measured following removal of the tissuefrom control or treated mice, flash-freezing on dry ice, homogenizationand sonication in distilled water, and pelleting of insolublemembranes/proteins by *centrifugation. Untreated, affected controls wereGAA-KO mice, 12 weeks old at the time of analysis except where notedotherwise. The protein concentrations of the clarified suspensions werequantified via the Bradford assay. hGAA activity in the muscle wasdetermined as described (Kikuchi, T., et al. (1998) J. Clin. Invest.101:827-833). Glycogen content of tissues was measured using theAspergillus niger assay system, as described (Amalfitano et al. (1999)Proc. Natl. Acad. Sci. USA 96:8861-8866). A two-tailed homoscedasticStudent's t-test was used to determine significant differences in hGAAlevels and glycogen content between GAA-KO mice with or withoutadministration of the vector encoding hGAA.

EXAMPLE 10 Western Blotting Analysis of hGAA

[0268] For direct detection of hGAA in tissues, samples (100 μg ofprotein) were electrophoresed overnight in a 6% polyacrylamide gel toseparate proteins, and transferred to a nylon membrane. The blots wereblocked with 5% nonfat milk solution, incubated with primary andsecondary antibodies and visualized via the enhanced chemiluminescence(ECL) detection system (Amersham Pharmacia, Piscataway, N.J.).

EXAMPLE 11 Results with Abbreviated hGAA cDNA

[0269] High-level production of hGAA in muscle was shown with an AAV2/6(AAV6) vector containing the shortened hGAA cDNA (FIG. 6). The hGAAlevel in skeletal muscle with the AAV6 vector is approximately 10-foldhigher than the GAA level in normal mice. The hGAA and glycogen contentwas analyzed in GAA-knockout (GAA-KO)/SCID mice that were treated at 6weeks of age, so these levels reflect the delivery of hGAA in adultanimals. The analysis was done 6 weeks following intramuscular AAV6injection, and demonstrated a trend toward long-term expression. Inaddition, glycogen content was significantly reduced (p<0.002),indicating a therapeutically-relevant effect from this level of hGAAproduction in muscle at that time point. Furthermore, prolonged hGAAexpression and complete reduction of glycogen content to normal wasobserved in the injected gastrocnemius muscle at 12 weeks after AAV6vector administration (not shown).

[0270] These data indicate that intramuscular injection of AAV vector isefficacious in glycogen storage disease II.

[0271] We have further shown secretion from liver with the AAV2/6 (AAV6)and AAV2/2 (AAV2) versions of the vector containing the shortened hGAAcDNA in GAA-KO/SCID mice following portal vein injection (FIG. 7). Theabbreviated GAA vector was packaged within either an AAV2 or AAV6 capsidto generate two different vector stocks. Each stock was administered toa different mouse by portal vein injection. hGAA was detected in plasmafrom both mice by Western blot analysis. The data further demonstrateuptake of hGAA by skeletal muscle and heart with these vectors.

[0272] These results indicate that the liver can be used as a depotorgan for hGAA production, with delivery to skeletal muscle and heartthat results in a reduction in glycogen stores in these tissues due touptake of secreted GAA.

EXAMPLE 12 Intramuscular Versus Intraportal Delivery of AAV Vectors inGAA-KO Mice

[0273] An AAV2 vector packaged as AAV1 (AAV2/1) corrected glycogenstorage when injected intramuscularly; however, the effect was observedonly in the injected muscle (Fraites et al. (2002) Mol. Ther.5:571-578). On the other hand, when we administered an AAV vector byportal vein injection in immunodeficient GAA-KO mice (GAA-KO/SCID mice),GAA was delivered to liver and other tissues (Sun et al. (2003) Mol.Ther. 2003; 7:467-477). Therefore, we investigated the difference inbenefit between intramuscular and intraportal injection of the AAVvector, since the latter approach delivered GAA to multiple targettissues including skeletal muscles, the diaphragm and the heart.

[0274] We chose to evaluate the benefit of an AAV vector encoding theshortened GAA targeted to skeletal muscle or to liver in the GAA-KO/SCIDmouse model. We administered an AAV vector encoding the shortened GM byintramuscular or portal vein injection in GAA-KO/SCID mice, and analyzedGAA activity and glycogen content in tissues. The AAV2-derived vectorwas packaged as AAV2 (AAV2/2) or AAV6, (AAV2/6) for portal veininjections. Based on homology between the capsid proteins of AAV6 andAAV1, it was deemed likely that AAV2/6 would transduce myofibers moreefficiently than AAV2/2 (Chao et al. (2000) Mol. Ther. 2:619-623;Rabinowitz et al. (2002) J. Virol. 76:791-801; Rutledge et al. (1998) J.Virol. 72:309-319). The AAV2/6 vector was injected intramuscularly.Glycogen content and GAA activity were analyzed in tissues at 6, 12, and24 Weeks after vector injection.

[0275] Results.

[0276] Following portal vein injection of an AAV2/2 vector, secreted GAAwas detectable by Western blot analysis of plasma starting at 2 weeksand persisted for 12 weeks (Data not shown). GAA activity was highlyelevated in multiple tissues compared to baseline levels in untreatedGAA-KO/SCID mice, and approximately 10-fold higher than wild-type levelsin the liver at 12 weeks following vector administration (FIG. 10).Human GAA was detected by Western blot analysis of multiple tissues,including the target tissues of heart and diaphragm (FIG. 11). Enzymeanalysis revealed that GAA activity was concordantly elevated in thesetissues (FIG. 10). These results demonstrated the secretion and uptakeof GAA following portal vein injection of the AAV vector, consistentwith the model of the liver as a depot organ for GAA production inGAA-deficient states as established with an Ad vector.

[0277] Following intramuscular injection with the AAV2/6 vector, GAAactivity in the gastrocnemius muscle exceeded normal levels byapproximately 20-fold at 6, 12 and 24 weeks following vectoradministration (FIG. 12). In these mice the glycogen content in theinjected muscle diminished to near-normal levels for up to 24 weeksfollowing vector administration, and decreased glycogen accumulation asevidenced by decreased staining for glycogen (FIG. 13). Despite theachievement of high-level GAA production in skeletal muscle, the GAAactivity in other tissues was not markedly increased (FIG. 12).

EXAMPLE 13 hGAA AAV Vectors with Altered Leader Signal Peptides

[0278] The peptide leader sequence (SEQ ID NO: 4, corresponding to aminoacids 1-27, SEQ ID NO: 2) of hGAA was replaced with the syntheticpeptide leader sequence SP38 (Barash et al., (2002) Biochem. Biophys.Res. Comm. 294:83542), as well as peptide leader sequences fromerythropoietin, albumin, alpha-1-antitrypsin and factor IX. The aminoacid sequences of these leader peptides are shown in Table 2. TABLE 2Amino acid sequences for leader peptides Leader sequence Peptidesequence hGAA MGVRHPPCSHRLLAVCALV (SEQ ID NO: 4) SLATAALL SP38MWWRLWWLLLLLLLLWPMV (SEQ ID NO: 5) WA Human MGVHECPAWLWLLLSLLSL (SEQ IDNO: 6) erythropoietin PLGLPVLG Human albumin MKWVTFISLLFLFSSAYS (SEQ IDNO: 7) Human alpha-i- MPSSVSWGILLLAGLCCLV (SEQ ID NO: 8) antitrypsinPVSLA Human coagula- MQRVNMIMAESPGLITICL (SEQ ID NO: 9) tion factor IXLGYLLSAECTVFLDHENAN KILNRPKR

[0279] Plasmid vectors that express hGAA in which the wild-type hGAApeptide leader sequence (SEQ ID NO: 4) is replaced with the differentleader peptides listed in Table 2 were constructed using the plasmidAVCBhGAAG-delta as described briefly below. The resulting constructsencode a GAA peptide in which one of the peptides of SEQ ID NOS: 5-9replaces the first 27 amino acids of SEQ ID NO: 1.

[0280] AVCBSP38GAAG-delta preparation strategy. A 0.37 kb PCR productfrom AVCBhGAAG-delta was amplified using the primers 5′-GCT GCA AAGCTTggg cac atc cta ctc cat-3′ (SEQ ID NO: 10) and 5′-cct gca gcc cct gctttg cag gga tgt agc-3′ (SEQ ID NO: 11). The resulting PCR productcomprises nucleotide sequences that code for the region of the hGAAprotein immediately downstream from the native hGAA signal peptide(nucleotides 523-796, SEQ ID NO: 1) and adds a HindIII restriction siteat the signal peptide cleavage site. This PCR product was digested withHindIII and SacII and gel-purified to produce DNA fragment 1.

[0281] A second DNA fragment coding for the SP38 leader sequence,KpnI-SP38-HindIII, 5′-AGC TGC TGA GGTACC TCA GCC A CC atg tgg tgg cgcctg tgg tgg ctg ctg ctg ctg ctg ctg ctg ctg tgg ccc atg gtg tgg gccAAGCTT CGA TGC TAC GTC-3′, SEQ ID NO: 12, was hybridized with a reverseprimer, 5′-GAC GTA GCA TCG MG CTT-₃′, SEQ ID NO: 13. The: resultinghybrid was extended Klenow DNA polymerase in the presence of dNTPs toform a double-stranded DNA fragment. The resulting DNA fragment containsa KpnI site near the 5′ end followed by an optimal Kozak sequence(GCCACC) and the SP38 leader peptide coding sequence, followed with aHindIII site at the 3′ end of the SP38 sequence. This DNA was digestedwith KpnI and HindIII and gel-purified to produce DNA fragment II.

[0282] DNA fragments I and II described above were then ligated intoAVCBhGAAG-delta plasmid DNA digested with KpnI and SacII. The resultingligation mixture was used to transform STBL2 cells (Gibco-BRL), andtransformants containing AVCBSP38GAAG-delta were selected in thepresence of antibiotics and structure confirmed by gel electrophoresis.The resulting construct codes for a GAA peptide in which the hGAA leadersequence (SEQ ID NO: 4) has been replaced with the SP38 leader sequence(SEQ ID NO: 5).

[0283] AVCBSP-hEpoGAAG-delta preparation strategy. The plasmidAVCBSP-hEpoGAAG-delta was prepared in the same manner asAVCBSP38GAAG-delta, wherein fragment II was replaced with theKpnI-HindIII digestion product of the double-stranded DNA fragment withsequence 5′-AGC TGC TGA GGTACC TCA GCC ACC atgggggtg cacgaatgtcctgcctggct gtggcttctc ctgtccctgc tgtcgctccc tctgggcctc ccagtcctgg gcAAGCTT CGA TGC TAC GTC-3′ (SEQ ID NO: 14). The resulting construct codesfor a GM peptide in which the hGAA leader sequence (SEQ ID NO: 4) hasbeen replaced with the hEPO leader sequence (SEQ ID NO: 6).

[0284] AVCBSPantitrypsinGAAG-delta preparation strategy. The plasmidAVCBSPantitrypsinGAAG-delta was prepared in the same manner asAVCBSP38GAAG-delta, using the KpnI-HindIII digestion product of thedouble-stranded DNA fragment with sequence 5′-C TGA GGTACC T GCC A CC-atgccgtcttct gtctcgtggg gcatcctcct gctggcaggc ctgtgctgcc tggtccctgtctccctggct-AAGCTT CGA T-3′ (SEQ ID NO: 15) in lieu of fragment II. Theresulting construct codes for a GAA peptide in which the hGAA leadersequence (SEQ ID NO: 4) has been replaced with the humanalpha-1-antityrpsin leader sequence (SEQ ID NO: 7).

[0285] AVCBSPALBGAAG-delta preparation strategy. AVCBSPALBGAAG-delta wasprepared in the same manner as AVCBSP38GAAG-delta, using theKpnI-HindIII digestion product of the double-stranded DNA fragment withsequence 5′-C TGA GGTACC T GCC ACC-a tgaagtgggt aacctttatt tcccttctttftctctttag ctcggcttat tcc-AAGCTT CGA T-3′ (SEQ ID NO: 16) in lieu offragment II. The resulting construct codes for a GAA peptide in whichthe hGAA leader sequence (SEQ ID NO: 4) has been replaced with the humanalbumin leader sequence (SEQ ID NO: 8).

[0286] AVCBSPFIXGAAG-delta preparation strategy. Lastly,AVCBSPFIXGAAG-delta was prepared in the same manner asAVCBSP38GAAG-delta, using the KpnI-HindIII digestion product of thedouble-stranded DNA fragment with sequence 5′-C TGA GGTACC T GCC ACCatgcagcgcg tgaacatgat catggca-3′ (SEQ ID NO: 17) in lieu of fragment II.The resulting construct codes for a GAA peptide in which the hGAA leadersequence (SEQ ID NO: 4) has been replaced with the human factor IXleader sequence (SEQ ID NO: 9).

[0287] The resulting plasmids were used to examine the effect ofdifferent leader sequences on the localization of hGAA in cellstransfected with these plasmids.

EXAMPLE 14 Relative Secretion of hGAA with Altered Leader Sequences in293 Cells

[0288] 293 cells were transfected with AAV vector plasmids described inExample 13 and were collected 40 hours post-transfection. Total hGAAactivity was assayed both in the cells and in the medium. These resultsare depicted in FIG. 14 and the relative hGAA secretion is summarized inTable 3. TABLE 3 hGAA secretion with different leader sequencesProportion hGAA % Total Increased Leader secreted (hGAA hGAA hGAA secre-sequence medium/hGAA cells) secreted tion (fold) HGAA 0.53 34 N/A SP38.19.7 91 18 Epo 1.1 53 2.1 α-1-antitrypsin 8.5 90 16 Factor IX 14 93 26

[0289] Western blot analysis of cellular and secreted hGAA in 293 cellstransfected with the AAV vector plasmids described in Example 13demonstrated normal migration, consistent with unaltered glycosylationand processing of chimeric hGAA linked to alternative signal peptides(FIG. 15). This data supported the normal glycosylation of hGAA, despitethe increased secretion and presumably shortened residence in the Golgi(Wisselaar et al., (1993) J. Biol. Chem 268 (3): 2223-31). For furtherevidence of the normal glycosylation of hGAA produced with theseconstructs, we injected mice with the AAV2/8 vector encoding thechimeric α-1-antitrypsin signal peptide linked to the hGAA cDNA (minusthe 27 amino acid GAA signal peptide), and hGAA was detectedcorresponding to the ˜110 kD hGAA precursor (FIG. 16). The hGAA levelwas lower for female mice (lanes 5-7 and 11-13), as expected given lowertransduction with AAV2/8 vectors in female mice. Significantly, hGAAsecretion was higher for the vector containing the chimeric α-1-antitrypsin signal peptide (lanes 2-7), than for the vector containingthe hGAA signal peptide (lanes 8-13).

[0290] Tissue GAA activity was increased in tissues for the 3 maleGAA-KO/SCID mice that received the AAV vector encoding thealpha-1-antitrypsin signal peptide linked to hGAA (corresponding tolanes 2-5 in FIG. 16). Significantly, liver GAA activity remained atnear normal levels, despite increased GAA activity in tissues comparedto controls (FIG. 17). These results were obtained at only 2 weeksfollowing vector administration, as opposed to the later 6-week timepoint used in the other experiments, and indicated early uptake ofsecreted GAA in the target tissues of heart, diaphragm, and at lowerlevels, skeletal muscle. GAA uptake in target tissues is expected toincrease after the 2-week time point, based on previous results.Finally, a Western blot of the target tissues showed appropriateprocessing of hGAA to ˜76 kD and ˜67 kD (data not shown). These datasupport the hypothesis that chimeric hGAA linked to alternative signalpeptides will be appropriately secreted, processed and targeted tolysosomes in target tissues, despite the maintenance of normal GAAlevels in liver. This novel development will reduce any potentialtoxicity for overexpression of hGAA in the depot organ, the liver, incontrast to all previous approaches to liver-targeted gene therapy inPompe disease that produced extreme supra-physiological levels of GAA inthe liver

EXAMPLE 15 Expression of hGAA with a Liver-Specific Promoter inImmunocompetent GAA-KO Mice

[0291] An AAV2/8 vector encoding hGAA driven by a liver-specificpromoter (Wang L et al., (1999) Proc Nat Acad Sci USA 96:3906-10) wasadministered intravenously in GAA-KO mice. Contrary to previousexperiments, where hGAA was eliminated in plasma by anti-GAA antibodiesin immunocompetent GAA-KO mice, hGAA persisted in plasma as detected byWestern blot analysis (FIG. 18). Therefore, the limitation of GAAexpression to the liver eliminated the antibody response and allowedpersistent GAA secretion with implications for gene therapy in Pompedisease.

[0292] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

1 17 1 3846 DNA Homo sapiens CDS (442)..(3300) 1 gcgcctgcgc gggaggccgcgtcacgtgac ccaccgcggc cccgccccgc gacgagctcc 60 cgccggtcac gtgacccgcctctgcgcgcc cccgggcacg accccggagt ctccgcgggc 120 ggccagggcg cgcgtgcgcggaggtgagcc gggccggggc tgcggggctt ccctgagcgc 180 gggccgggtc ggtggggcggtcggctgccc gcgccggcct ctcagttggg aaagctgagg 240 ttgtcgccgg ggccgcgggtggaggtcggg gatgaggcag caggtaggac agtgacctcg 300 gtgacgcgaa ggaccccggccacctctagg ttctcctcgt ccgcccgttg ttcagcgagg 360 gaggctctgg gcctgccgcagctgacgggg aaactgaggc acggagcggg cctgtaggag 420 ctgtccaggc catctccaac catg gga gtg agg cac ccg ccc tgc tcc cac 471 Met Gly Val Arg His Pro ProCys Ser His 1 5 10 cgg ctc ctg gcc gtc tgc gcc ctc gtg tcc ttg gca accgct gca ctc 519 Arg Leu Leu Ala Val Cys Ala Leu Val Ser Leu Ala Thr AlaAla Leu 15 20 25 ctg ggg cac atc cta ctc cat gat ttc ctg ctg gtt ccc cgagag ctg 567 Leu Gly His Ile Leu Leu His Asp Phe Leu Leu Val Pro Arg GluLeu 30 35 40 agt ggc tcc tcc cca gtc ctg gag gag act cac cca gct cac cagcag 615 Ser Gly Ser Ser Pro Val Leu Glu Glu Thr His Pro Ala His Gln Gln45 50 55 gga gcc agc aga cca ggg ccc cgg gat gcc cag gca cac ccc ggc cgt663 Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro Gly Arg 6065 70 ccc aga gca gtg ccc aca cag tgc gac gtc ccc ccc aac agc cgc ttc711 Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser Arg Phe 7580 85 90 gat tgc gcc cct gac aag gcc atc acc cag gaa cag tgc gag gcc cgc759 Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg 95100 105 ggc tgc tgc tac atc cct gca aag cag ggg ctg cag gga gcc cag atg807 Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala Gln Met 110115 120 ggg cag ccc tgg tgc ttc ttc cca ccc agc tac ccc agc tac aag ctg855 Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu 125130 135 gag aac ctg agc tcc tct gaa atg ggc tac acg gcc acc ctg acc cgt903 Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg 140145 150 acc acc ccc acc ttc ttc ccc aag gac atc ctg acc ctg cgg ctg gac951 Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp 155160 165 170 gtg atg atg gag act gag aac cgc ctc cac ttc acg atc aaa gatcca 999 Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys Asp Pro175 180 185 gct aac agg cgc tac gag gtg ccc ttg gag acc ccg cgt gtc cacagc 1047 Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg Val His Ser190 195 200 cgg gca ccg tcc cca ctc tac agc gtg gag ttc tcc gag gag cccttc 1095 Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe205 210 215 ggg gtg atc gtg cac cgg cag ctg gac ggc cgc gtg ctg ctg aacacg 1143 Gly Val Ile Val His Arg Gln Leu Asp Gly Arg Val Leu Leu Asn Thr220 225 230 acg gtg gcg ccc ctg ttc ttt gcg gac cag ttc ctt cag ctg tccacc 1191 Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr235 240 245 250 tcg ctg ccc tcg cag tat atc aca ggc ctc gcc gag cac ctcagt ccc 1239 Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu SerPro 255 260 265 ctg atg ctc agc acc agc tgg acc agg atc acc ctg tgg aaccgg gac 1287 Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn ArgAsp 270 275 280 ctt gcg ccc acg ccc ggt gcg aac ctc tac ggg tct cac cctttc tac 1335 Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro PheTyr 285 290 295 ctg gcg ctg gag gac ggc ggg tcg gca cac ggg gtg ttc ctgcta aac 1383 Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu LeuAsn 300 305 310 agc aat gcc atg gat gtg gtc ctg cag ccg agc cct gcc cttagc tgg 1431 Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu SerTrp 315 320 325 330 agg tcg aca ggt ggg atc ctg gat gtc tac atc ttc ctgggc cca gag 1479 Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu GlyPro Glu 335 340 345 ccc aag agc gtg gtg cag cag tac ctg gac gtt gtg ggatac ccg ttc 1527 Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly TyrPro Phe 350 355 360 atg ccg cca tac tgg ggc ctg ggc ttc cac ctg tgc cgctgg ggc tac 1575 Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg TrpGly Tyr 365 370 375 tcc tcc acc gct atc acc cgc cag gtg gtg gag aac atgacc agg gcc 1623 Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met ThrArg Ala 380 385 390 cac ttc ccc ctg gac gtc caa tgg aac gac ctg gac tacatg gac tcc 1671 His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr MetAsp Ser 395 400 405 410 cgg agg gac ttc acg ttc aac aag gat ggc ttc cgggac ttc ccg gcc 1719 Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg AspPhe Pro Ala 415 420 425 atg gtg cag gag ctg cac cag ggc ggc cgg cgc tacatg atg atc gtg 1767 Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr MetMet Ile Val 430 435 440 gat cct gcc atc agc agc tcg ggc cct gcc ggg agctac agg ccc tac 1815 Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser TyrArg Pro Tyr 445 450 455 gac gag ggt ctg cgg agg ggg gtt ttc atc acc aacgag acc ggc cag 1863 Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn GluThr Gly Gln 460 465 470 ccg ctg att ggg aag gta tgg ccc ggg tcc act gccttc ccc gac ttc 1911 Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala PhePro Asp Phe 475 480 485 490 acc aac ccc aca gcc ctg gcc tgg tgg gag gacatg gtg gct gag ttc 1959 Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp MetVal Ala Glu Phe 495 500 505 cat gac cag gtg ccc ttc gac ggc atg tgg attgac atg aac gag cct 2007 His Asp Gln Val Pro Phe Asp Gly Met Trp Ile AspMet Asn Glu Pro 510 515 520 tcc aac ttc atc aga ggc tct gag gac ggc tgcccc aac aat gag ctg 2055 Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys ProAsn Asn Glu Leu 525 530 535 gag aac cca ccc tac gtg cct ggg gtg gtt gggggg acc ctc cag gcg 2103 Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly GlyThr Leu Gln Ala 540 545 550 gcc acc atc tgt gcc tcc agc cac cag ttt ctctcc aca cac tac aac 2151 Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu SerThr His Tyr Asn 555 560 565 570 ctg cac aac ctc tac ggc ctg acc gaa gccatc gcc tcc cac agg gcg 2199 Leu His Asn Leu Tyr Gly Leu Thr Glu Ala IleAla Ser His Arg Ala 575 580 585 ctg gtg aag gct cgg ggg aca cgc cca tttgtg atc tcc cgc tcg acc 2247 Leu Val Lys Ala Arg Gly Thr Arg Pro Phe ValIle Ser Arg Ser Thr 590 595 600 ttt gct ggc cac ggc cga tac gcc ggc cactgg acg ggg gac gtg tgg 2295 Phe Ala Gly His Gly Arg Tyr Ala Gly His TrpThr Gly Asp Val Trp 605 610 615 agc tcc tgg gag cag ctc gcc tcc tcc gtgcca gaa atc ctg cag ttt 2343 Ser Ser Trp Glu Gln Leu Ala Ser Ser Val ProGlu Ile Leu Gln Phe 620 625 630 aac ctg ctg ggg gtg cct ctg gtc ggg gccgac gtc tgc ggc ttc ctg 2391 Asn Leu Leu Gly Val Pro Leu Val Gly Ala AspVal Cys Gly Phe Leu 635 640 645 650 ggc aac acc tca gag gag ctg tgt gtgcgc tgg acc cag ctg ggg gcc 2439 Gly Asn Thr Ser Glu Glu Leu Cys Val ArgTrp Thr Gln Leu Gly Ala 655 660 665 ttc tac ccc ttc atg cgg aac cac aacagc ctg ctc agt ctg ccc cag 2487 Phe Tyr Pro Phe Met Arg Asn His Asn SerLeu Leu Ser Leu Pro Gln 670 675 680 gag ccg tac agc ttc agc gag ccg gcccag cag gcc atg agg aag gcc 2535 Glu Pro Tyr Ser Phe Ser Glu Pro Ala GlnGln Ala Met Arg Lys Ala 685 690 695 ctc acc ctg cgc tac gca ctc ctc ccccac ctc tac aca ctg ttc cac 2583 Leu Thr Leu Arg Tyr Ala Leu Leu Pro HisLeu Tyr Thr Leu Phe His 700 705 710 cag gcc cac gtc gcg ggg gag acc gtggcc cgg ccc ctc ttc ctg gag 2631 Gln Ala His Val Ala Gly Glu Thr Val AlaArg Pro Leu Phe Leu Glu 715 720 725 730 ttc ccc aag gac tct agc acc tggact gtg gac cac cag ctc ctg tgg 2679 Phe Pro Lys Asp Ser Ser Thr Trp ThrVal Asp His Gln Leu Leu Trp 735 740 745 ggg gag gcc ctg ctc atc acc ccagtg ctc cag gcc ggg aag gcc gaa 2727 Gly Glu Ala Leu Leu Ile Thr Pro ValLeu Gln Ala Gly Lys Ala Glu 750 755 760 gtg act ggc tac ttc ccc ttg ggcaca tgg tac gac ctg cag acg gtg 2775 Val Thr Gly Tyr Phe Pro Leu Gly ThrTrp Tyr Asp Leu Gln Thr Val 765 770 775 cca ata gag gcc ctt ggc agc ctccca ccc cca cct gca gct ccc cgt 2823 Pro Ile Glu Ala Leu Gly Ser Leu ProPro Pro Pro Ala Ala Pro Arg 780 785 790 gag cca gcc atc cac agc gag gggcag tgg gtg acg ctg ccg gcc ccc 2871 Glu Pro Ala Ile His Ser Glu Gly GlnTrp Val Thr Leu Pro Ala Pro 795 800 805 810 ctg gac acc atc aac gtc cacctc cgg gct ggg tac atc atc ccc ctg 2919 Leu Asp Thr Ile Asn Val His LeuArg Ala Gly Tyr Ile Ile Pro Leu 815 820 825 cag ggc cct ggc ctc aca accaca gag tcc cgc cag cag ccc atg gcc 2967 Gln Gly Pro Gly Leu Thr Thr ThrGlu Ser Arg Gln Gln Pro Met Ala 830 835 840 ctg gct gtg gcc ctg acc aagggt gga gag gcc cga ggg gag ctg ttc 3015 Leu Ala Val Ala Leu Thr Lys GlyGly Glu Ala Arg Gly Glu Leu Phe 845 850 855 tgg gac gat gga gag agc ctggaa gtg ctg gag cga ggg gcc tac aca 3063 Trp Asp Asp Gly Glu Ser Leu GluVal Leu Glu Arg Gly Ala Tyr Thr 860 865 870 cag gtc atc ttc ctg gcc aggaat aac acg atc gtg aat gag ctg gta 3111 Gln Val Ile Phe Leu Ala Arg AsnAsn Thr Ile Val Asn Glu Leu Val 875 880 885 890 cgt gtg acc agt gag ggagct ggc ctg cag ctg cag aag gtg act gtc 3159 Arg Val Thr Ser Glu Gly AlaGly Leu Gln Leu Gln Lys Val Thr Val 895 900 905 ctg ggc gtg gcc acg gcgccc cag cag gtc ctc tcc aac ggt gtc cct 3207 Leu Gly Val Ala Thr Ala ProGln Gln Val Leu Ser Asn Gly Val Pro 910 915 920 gtc tcc aac ttc acc tacagc ccc gac acc aag gtc ctg gac atc tgt 3255 Val Ser Asn Phe Thr Tyr SerPro Asp Thr Lys Val Leu Asp Ile Cys 925 930 935 gtc tcg ctg ttg atg ggagag cag ttt ctc gtc agc tgg tgt tag 3300 Val Ser Leu Leu Met Gly Glu GlnPhe Leu Val Ser Trp Cys 940 945 950 ccgggcggag tgtgttagtc tctccagagggaggctggtt ccccagggaa gcagagcctg 3360 tgtgcgggca gcagctgtgt gcgggcctgggggttgcatg tgtcacctgg agctgggcac 3420 taaccattcc aagccgccgc atcgcttgtttccacctcct gggccggggc tctggccccc 3480 aacgtgtcta ggagagcttt ctccctagatcgcactgtgg gccggggcct ggagggctgc 3540 tctgtgttaa taagattgta aggtttgccctcctcacctg ttgccggcat gcgggtagta 3600 ttagccaccc ccctccatct gttcccagcaccggagaagg gggtgctcag gtggaggtgt 3660 ggggtatgca cctgagctcc tgcttcgcgcctgctgctct gccccaacgc gaccgcttcc 3720 cggctgccca gagggctgga tgcctgccggtccccgagca agcctgggaa ctcaggaaaa 3780 ttcacaggac ttgggagatt ctaaatcttaagtgcaatta ttttaataaa aggggcattt 3840 ggaatc 3846 2 952 PRT Homo sapiens2 Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys 1 5 1015 Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu 20 2530 His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val 35 4045 Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly 50 5560 Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr 65 7075 80 Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 8590 95 Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro100 105 110 Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp CysPhe 115 120 125 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu SerSer Ser 130 135 140 Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr ProThr Phe Phe 145 150 155 160 Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp ValMet Met Glu Thr Glu 165 170 175 Asn Arg Leu His Phe Thr Ile Lys Asp ProAla Asn Arg Arg Tyr Glu 180 185 190 Val Pro Leu Glu Thr Pro Arg Val HisSer Arg Ala Pro Ser Pro Leu 195 200 205 Tyr Ser Val Glu Phe Ser Glu GluPro Phe Gly Val Ile Val His Arg 210 215 220 Gln Leu Asp Gly Arg Val LeuLeu Asn Thr Thr Val Ala Pro Leu Phe 225 230 235 240 Phe Ala Asp Gln PheLeu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr 245 250 255 Ile Thr Gly LeuAla Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser 260 265 270 Trp Thr ArgIle Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly 275 280 285 Ala AsnLeu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly 290 295 300 GlySer Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val 305 310 315320 Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 325330 335 Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln340 345 350 Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr TrpGly 355 360 365 Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr AlaIle Thr 370 375 380 Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe ProLeu Asp Val 385 390 395 400 Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser ArgArg Asp Phe Thr Phe 405 410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro AlaMet Val Gln Glu Leu His 420 425 430 Gln Gly Gly Arg Arg Tyr Met Met IleVal Asp Pro Ala Ile Ser Ser 435 440 445 Ser Gly Pro Ala Gly Ser Tyr ArgPro Tyr Asp Glu Gly Leu Arg Arg 450 455 460 Gly Val Phe Ile Thr Asn GluThr Gly Gln Pro Leu Ile Gly Lys Val 465 470 475 480 Trp Pro Gly Ser ThrAla Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp GluAsp Met Val Ala Glu Phe His Asp Gln Val Pro Phe 500 505 510 Asp Gly MetTrp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly 515 520 525 Ser GluAsp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val 530 535 540 ProGly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser 545 550 555560 Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly 565570 575 Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly580 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His GlyArg 595 600 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp GluGln Leu 610 615 620 Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu LeuGly Val Pro 625 630 635 640 Leu Val Gly Ala Asp Val Cys Gly Phe Leu GlyAsn Thr Ser Glu Glu 645 650 655 Leu Cys Val Arg Trp Thr Gln Leu Gly AlaPhe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu Leu Ser Leu ProGln Glu Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala Gln Gln Ala Met ArgLys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr ThrLeu Phe His Gln Ala His Val Ala Gly 705 710 715 720 Glu Thr Val Ala ArgPro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser 725 730 735 Thr Trp Thr ValAsp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro ValLeu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu GlyThr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly 770 775 780 SerLeu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser 785 790 795800 Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val 805810 815 His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr820 825 830 Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala LeuThr 835 840 845 Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp GlyGlu Ser 850 855 860 Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val IlePhe Leu Ala 865 870 875 880 Arg Asn Asn Thr Ile Val Asn Glu Leu Val ArgVal Thr Ser Glu Gly 885 890 895 Ala Gly Leu Gln Leu Gln Lys Val Thr ValLeu Gly Val Ala Thr Ala 900 905 910 Pro Gln Gln Val Leu Ser Asn Gly ValPro Val Ser Asn Phe Thr Tyr 915 920 925 Ser Pro Asp Thr Lys Val Leu AspIle Cys Val Ser Leu Leu Met Gly 930 935 940 Glu Gln Phe Leu Val Ser TrpCys 945 950 3 3026 DNA Homo sapiens 3 gcctgtagga gctgtccagg ccatctccaaccatgggagt gaggcacccg ccctgctccc 60 accggctcct ggccgtctgc gccctcgtgtccttggcaac cgctgcactc ctggggcaca 120 tcctactcca tgatttcctg ctggttccccgagagctgag tggctcctcc ccagtcctgg 180 aggagactca cccagctcac cagcagggagccagcagacc agggccccgg gatgcccagg 240 cacaccccgg ccgtcccaga gcagtgcccacacagtgcga cgtccccccc aacagccgct 300 tcgattgcgc ccctgacaag gccatcacccaggaacagtg cgaggcccgc ggctgctgct 360 acatccctgc aaagcagggg ctgcagggagcccagatggg gcagccctgg tgcttcttcc 420 cacccagcta ccccagctac aagctggagaacctgagctc ctctgaaatg ggctacacgg 480 ccaccctgac ccgtaccacc cccaccttcttccccaagga catcctgacc ctgcggctgg 540 acgtgatgat ggagactgag aaccgcctccacttcacgat caaagatcca gctaacaggc 600 gctacgaggt gcccttggag accccgcgtgtccacagccg ggcaccgtcc ccactctaca 660 gcgtggagtt ctccgaggag cccttcggggtgatcgtgca ccggcagctg gacggccgcg 720 tgctgctgaa cacgacggtg gcgcccctgttctttgcgga ccagttcctt cagctgtcca 780 cctcgctgcc ctcgcagtat atcacaggcctcgccgagca cctcagtccc ctgatgctca 840 gcaccagctg gaccaggatc accctgtggaaccgggacct tgcgcccacg cccggtgcga 900 acctctacgg gtctcaccct ttctacctggcgctggagga cggcgggtcg gcacacgggg 960 tgttcctgct aaacagcaat gccatggatgtggtcctgca gccgagccct gcccttagct 1020 ggaggtcgac aggtgggatc ctggatgtctacatcttcct gggcccagag cccaagagcg 1080 tggtgcagca gtacctggac gttgtgggatacccgttcat gccgccatac tggggcctgg 1140 gcttccacct gtgccgctgg ggctactcctccaccgctat cacccgccag gtggtggaga 1200 acatgaccag ggcccacttc cccctggacgtccaatggaa cgacctggac tacatggact 1260 cccggaggga cttcacgttc aacaaggatggcttccggga cttcccggcc atggtgcagg 1320 agctgcacca gggcggccgg cgctacatgatgatcgtgga tcctgccatc agcagctcgg 1380 gccctgccgg gagctacagg ccctacgacgagggtctgcg gaggggggtt ttcatcacca 1440 acgagaccgg ccagccgctg attgggaaggtatggcccgg gtccactgcc ttccccgact 1500 tcaccaaccc cacagccctg gcctggtgggaggacatggt ggctgagttc catgaccagg 1560 tgcccttcga cggcatgtgg attgacatgaacgagccttc caacttcatc agaggctctg 1620 aggacggctg ccccaacaat gagctggagaacccacccta cgtgcctggg gtggttgggg 1680 ggaccctcca ggcggccacc atctgtgcctccagccacca gtttctctcc acacactaca 1740 acctgcacaa cctctacggc ctgaccgaagccatcgcctc ccacagggcg ctggtgaagg 1800 ctcgggggac acgcccattt gtgatctcccgctcgacctt tgctggccac ggccgatacg 1860 ccggccactg gacgggggac gtgtggagctcctgggagca gctcgcctcc tccgtgccag 1920 aaatcctgca gtttaacctg ctgggggtgcctctggtcgg ggccgacgtc tgcggcttcc 1980 tgggcaacac ctcagaggag ctgtgtgtgcgctggaccca gctgggggcc ttctacccct 2040 tcatgcggaa ccacaacagc ctgctcagtctgccccagga gccgtacagc ttcagcgagc 2100 cggcccagca ggccatgagg aaggccctcaccctgcgcta cgcactcctc ccccacctct 2160 acacactgtt ccaccaggcc cacgtcgcgggggagaccgt ggcccggccc ctcttcctgg 2220 agttccccaa ggactctagc acctggactgtggaccacca gctcctgtgg ggggaggccc 2280 tgctcatcac cccagtgctc caggccgggaaggccgaagt gactggctac ttccccttgg 2340 gcacatggta cgacctgcag acggtgccaatagaggccct tggcagcctc ccacccccac 2400 ctgcagctcc ccgtgagcca gccatccacagcgaggggca gtgggtgacg ctgccggccc 2460 ccctggacac catcaacgtc cacctccgggctgggtacat catccccctg cagggccctg 2520 gcctcacaac cacagagtcc cgccagcagcccatggccct ggctgtggcc ctgaccaagg 2580 gtggagaggc ccgaggggag ctgttctgggacgatggaga gagcctggaa gtgctggagc 2640 gaggggccta cacacaggtc atcttcctggccaggaataa cacgatcgtg aatgagctgg 2700 tacgtgtgac cagtgaggga gctggcctgcagctgcagaa ggtgactgtc ctgggcgtgg 2760 ccacggcgcc ccagcaggtc ctctccaacggtgtccctgt ctccaacttc acctacagcc 2820 ccgacaccaa ggtcctggac atctgtgtctcgctgttgat gggagagcag tttctcgtca 2880 gctggtgtta gccgggcgga gtgtgttagtctctccagag ggaggctggt tccccaggga 2940 agcagagcct gtgtgcgggc agcagctgtgtgcgggcctg ggggttgtta agtgcaatta 3000 ttttaataaa aggggcattt ggaatc 30264 27 PRT Homo sapiens 4 Met Gly Val Arg His Pro Pro Cys Ser His Arg LeuLeu Ala Val Cys 1 5 10 15 Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu 2025 5 21 PRT Artificial Sequence Artificial secretory signal 5 Met TrpTrp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 ProMet Val Trp Ala 20 6 27 PRT Homo sapiens 6 Met Gly Val His Glu Cys ProAla Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu GlyLeu Pro Val Leu Gly 20 25 7 18 PRT Homo sapiens 7 Met Lys Trp Val ThrPhe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser 8 24 PRTHomo sapiens 8 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala GlyLeu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala 20 9 46 PRT Homosapiens 9 Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu IleThr 1 5 10 15 Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr ValPhe Leu 20 25 30 Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg35 40 45 10 30 DNA Artificial Sequence Synthetic oligonucleotide primer10 gctgcaaagc ttgggcacat cctactccat 30 11 30 DNA Artificial SequenceSynthetic oligonucleotide primer 11 cctgcagccc ctgctttgca gggatgtagc 3012 105 DNA Artificial Sequence Synthetic oligonucleotide 12 agctgctgaggtacctcagc caccatgtgg tggcgcctgt ggtggctgct gctgctgctg 60 ctgctgctgtggcccatggt gtgggccaag cttcgatgct acgtc 105 13 18 DNA Artificial SequenceSynthetic oligonucleotide primer 13 gacgtagcat cgaagctt 18 14 123 DNAArtificial Sequence Synthetic oligonucleotide 14 agctgctgag gtacctcagccaccatgggg gtgcacgaat gtcctgcctg gctgtggctt 60 ctcctgtccc tgctgtcgctccctctgggc ctcccagtcc tgggcaagct tcgatgctac 120 gtc 123 15 99 DNAArtificial Sequence Synthetic oligonucleotide 15 ctgaggtacc tgccaccatgccgtcttctg tctcgtgggg catcctcctg ctggcaggcc 60 tgtgctgcct ggtccctgtctccctggcta agcttcgat 99 16 81 DNA Artificial Sequence Syntheticoligonucleotide 16 ctgaggtacc tgccaccatg aagtgggtaa cctttatttcccttcttttt ctctttagct 60 cggcttattc caagcttcga t 81 17 44 DNA ArtificialSequence Synthetic oligonucleotide 17 ctgaggtacc tgccaccatg cagcgcgtgaacatgatcat ggca 44

That which is claimed is:
 1. An isolated nucleic acid encoding achimeric polypeptide comprising a secretory signal sequence operablylinked to a lysosomal polypeptide.
 2. The isolated nucleic acid of claim1, wherein said secretory signal sequence is derived from a, secretedpolypeptide.
 3. The isolated nucleic acid of claim 2, wherein saidsecretory signal sequence is derived from erythropoietin, Factor IX,prealbumin or α1-antitrypsin precursor polypeptide.
 4. The isolatednucleic acid of claim 1, wherein said secretory signal sequencecomprises the amino acid sequence of SEQ ID NO:5.
 5. The isolatednucleic acid of claim 1, wherein said isolated nucleic acid isoperatively linked to a transcriptional control element operable, inliver cells.
 6. The isolated nucleic acid of claim 1, wherein saidlysosomal polypeptide is a lysosomal acid α-glucosidase (GAA)polypeptide.
 7. The isolated nucleic acid of claim 6, wherein said GAApolypeptide is a human GAA polypeptide.
 8. The isolated nucleic acid ofclaim 6, wherein said isolated nucleic acid further comprises a 3′untranslated region.
 9. The isolated nucleic acid of claim 8, wherein:(a) said 3′ untranslated region comprises a deletion therein; or (b)wherein said 3′ untranslated region is less than 200 nucleotides inlength and comprises a segment that is heterologous to said GAA codingregion.
 10. The isolated nucleic acid of claim 1, wherein the isolatednucleic acid is 4 kilobases or less in length.
 11. A vector comprisingthe isolated nucleic acid of claim
 1. 12. The vector of claim 11,wherein said vector is an adeno-associated virus (AAV) vector.
 13. Thevector of claim 11, wherein said lysosomal polypeptide is a GAApolypeptide.
 14. A pharmaceutical formulation comprising the isolatednucleic acid of claim 1 in a pharmaceutically acceptable carrier. 15.The pharmaceutical formulation of claim 14, wherein said pharmaceuticalformulation comprises a vector comprising the isolated nucleic acid. 16.The pharmaceutical formulation of claim 15, wherein said vector is anadeno-associated virus (AAV) vector.
 17. The pharmaceutical formulationof claim 14, wherein said lysosomal polypeptide is a GAA polypeptide.18. A cell comprising the isolated nucleic acid of claim
 1. 19. Achimeric polypeptide comprising a secretory signal sequence operablylinked to a lysosomal polypeptide.
 20. The chimeric polypeptide of claim19, wherein said lysosomal polypeptide is an acid α-glucosidase (GAA)polypeptide.
 21. A method of delivering a nucleic acid encoding alysosomal polypeptide to a cell, comprising contacting a cell with anisolated nucleic acid according to claim 1 under conditions sufficientfor the isolated nucleic acid to be introduced into the cell, expressedto produce the chimeric polypeptide comprising the secretory signalsequence operably linked to the lysosomal polypeptide, and the lysosomalpolypeptide secreted from the cell.
 22. The method of claim 21, whereinthe cell is contacted with a vector comprising the isolated nucleicacid.
 23. The method of claim 21, wherein said lysosomal polypeptide isa lysosomal acid α-glucosidase (GAA) polypeptide.
 24. The method ofclaim 21, wherein the cell is a cultured cell.
 25. The method of claim21, wherein the cell is a cell in vivo.
 26. A method of producing alysosomal acid α-glucosidase (GAA) polypeptide in a cultured cell,comprising: contacting a cultured cell with an isolated nucleic acidaccording to claim 6 under conditions sufficient for the isolatednucleic acid to be introduced into the cultured cell, expressed toproduce the chimeric polypeptide comprising the secretory signalsequence operably linked to the GAA polypeptide, and the GAA polypeptidesecreted from the cultured cell, and collecting the GAA polypeptidesecreted into the cell culture medium.
 27. The method of claim 26,wherein the cell is a mammalian cell.
 28. The method of claim 27,wherein the cell is a CHO cell, a 293 cell, a HT1080 cell, a HeLa cellor a C10 cell.
 29. The method of claim 26, wherein the cell is contactedwith a vector comprising the isolated nucleic acid.
 30. A method oftreating a deficiency of a lysosomal polypeptide in a subject,comprising administering to the subject a cell according to claim 18 ina pharmaceutically acceptable carrier in a therapeutically effectiveamount.
 31. A method of treating a deficiency of a lysosomal polypeptidein a subject, comprising administering to the subject the pharmaceuticalformulation of claim 14 in a therapeutically effective amount.
 32. Themethod of claim 31, wherein the pharmaceutical formulation comprises avector comprising the isolated nucleic acid.
 33. The method of claim 31,wherein the isolated nucleic acid encoding the chimeric polypeptide isdelivered to the liver.
 34. A method of treating a deficiency of alysosomal polypeptide in a subject, comprising administering to thesubject the pharmaceutical formulation of claim 17 in a therapeuticallyeffective amount.
 35. The method of claim 34, wherein the pharmaceuticalformulation comprises a vector comprising the isolated nucleic acid. 36.The method of claim 34, wherein the isolated nucleic acid encoding thechimeric polypeptide is delivered to the liver.
 37. The method of claim36, wherein the GAA polypeptide is secreted from the liver and there isuptake of the secreted GAA polypeptide by skeletal muscle tissue,cardiac muscle tissue, diaphragm muscle tissue or a combination thereof,wherein uptake of the secreted GAA polypeptide results in a reduction inlysosomal glycogen stores in the tissue(s).
 38. An isolated nucleic acidencoding a lysosomal acid α-glucosidase (GAA) polypeptide, said isolatednucleic acid comprising: (a) a coding region encoding a GAA polypeptide,and (b) a 3′ untranslated region, (i) wherein said 3′ untranslatedregion comprises a GAA 3′ untranslated region comprising a deletion ofat least 25 consecutive nucleotides, so that upon introduction into acell, GAA polypeptide is produced at a higher level from said isolatednucleic acid as compared with GAA polypeptide production from anisolated nucleic acid comprising a full-length GAA 3′ untranslatedregion, or (ii) wherein said 3′ untranslated region is less than 200nucleotides in length and comprises a segment that is heterologous tosaid GAA coding region, so that upon introduction into a cell, GAApolypeptide is produced at a higher level from said isolated nucleicacid as compared with GAA polypeptide production from an isolatednucleic acid comprising a full-length GAA 3′ untranslated region. 39.The isolated nucleic acid of claim 38, wherein said 3′ untranslatedregion comprises the deletion of subparagraph (i).
 40. The isolatednucleic acid of claim 39, wherein said deletion in said 3′ untranslatedregion comprises a deletion of at least 100 bases.
 41. The isolatednucleic acid of claim 39, wherein at least 50% of said 3′ untranslatedregion has been deleted.
 42. The isolated nucleic acid of claim 39,wherein said 3′ untranslated region is 200 nucleotides in length orless.
 43. The isolated nucleic acid of claim 42, wherein essentially allof said 3′ untranslated region has been deleted.
 44. The isolatednucleic acid of claim 39, wherein said 3′ untranslated region comprisesa deleted form of the 3′ untranslated region at nucleotides 3301 to 3846of SEQ ID NO:1.
 45. The isolated nucleic acid of claim 44, wherein said3′ untranslated region comprises nucleotides 2878 to 3012 of SEQ IDNO:3.
 46. The isolated nucleic acid of claim 39, wherein said isolatednucleic acid comprises SEQ ID NO:3.
 47. The isolated nucleic acid ofclaim 38, wherein said GAA polypeptide is a human GAA polypeptide. 48.The isolated nucleic acid of claim 38, wherein said isolated nucleicacid is operatively linked to a transcriptional control element that isoperable in liver cells.
 49. The isolated nucleic acid of claim 38,wherein said isolated nucleic acid is 4 kilobases or less in length. 50.A vector comprising the isolated nucleic acid of claim
 38. 51. Thevector of claim 50, wherein said vector is an adeno-associated virus(AAV) vector.
 52. A pharmaceutical formulation comprising the isolatednucleic acid of claim 38 in a pharmaceutically acceptable carrier. 53.The pharmaceutical formulation of claim 52, wherein said pharmaceuticalformulation comprises a vector comprising the isolated nucleic acid. 54.A cell comprising the isolated nucleic acid of claim
 38. 55. A method ofdelivering a nucleic acid encoding a lysosomal acid α-glucosidase (GAA)polypeptide to a cell, comprising contacting a cell with the isolatednucleic acid according to claim 38 under conditions sufficient for theisolated nucleic acid encoding the GAA polypeptide to be introduced intothe cell and expressed to produce the GAA polypeptide.
 56. The method ofclaim 55, wherein the cell is contacted with a vector comprising theisolated nucleic acid encoding the GAA polypeptide.
 57. The method ofclaim 56, wherein the vector is an adeno-associated virus (AAV) vector.58. The method of claim 55, wherein the cell is a cultured cell.
 59. Amethod of producing lysosomal acid α-glucosidase (GAA) polypeptide in acultured cell, comprising: contacting a cultured cell with an isolatednucleic acid according to claim 38 under conditions sufficient for theisolated nucleic acid to be introduced into the cultured cell andexpressed to produce the GAA polypeptide, and collecting the GAApolypeptide.
 60. The method of claim 59, wherein the GAA polypeptide issecreted into the cell culture medium and collected therefrom.
 61. Themethod of claim 59, wherein the cell is a mammalian cell.
 62. The methodof claim 62, wherein the cell is a CHO cell, a 293 cell, a HT1080 cell,a HeLa cell or a C10 cell.
 63. The method of claim 59, wherein the cellis contacted with a vector comprising the isolated nucleic acid.
 64. Themethod of claim 63, wherein the vector is an adeno-associated virus(AAV) vector.
 65. A method of treating lysosomal acid α-glucosidase(GAA) deficiency in a subject, comprising administering to the subject acell according to claim 54 in a pharmaceutically acceptable carrier in atherapeutically effective amount.
 66. A method of treating lysosomalacid α-glucosidase (GAA) deficiency in a subject, comprisingadministering to the subject the pharmaceutical formulation of claim 52in a therapeutically effective amount.
 67. The method of claim 66,wherein the subject is a human subject.
 68. The method of claim 66,wherein the isolated nucleic acid encoding GAA is delivered to theliver.
 69. The method of claim 68, wherein the GAA polypeptide issecreted from the liver into the systemic circulation.
 70. The method ofclaim 69, wherein there is uptake of the secreted GAA polypeptide byskeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissueor a combination thereof, wherein uptake of the secreted GAA polypeptideresults in a reduction in lysosomal glycogen stores in the tissue(s).71. The method of claim 66, wherein the pharmaceutical formulationcomprises a vector comprising-the isolated nucleic acid encoding the GAApolypeptide.
 72. The method of claim 71, wherein the vector is anadeno-associated virus (AAV) vector.